Chikungunya virus (CHIKV) was introduced to the Americas in 2013, causing two million infections across over thirty countries. CHIKV causes a chronic debilitating arthritis in one fourth of infected individuals and currently evidence-based targeted therapies for the treatment of CHIKV arthritis are lacking. Multiple mouse models of chikungunya have been developed to study acute CHIKV infection. In humans, post-CHIKV arthritis may persist for months to years after viremia from a CHIKV infection has resolved. Therefore, the development of a mouse model of post-acute arthritis of chikungunya may facilitate the study of potential novel therapeutics for this arthritis. In this article we describe the development of a wild-type immunocompetent C57BL/6 mouse model for post-acute arthritis of chikungunya, including a histologic inflammation scoring system, as well as suggestions for how this mouse model may be used to examine the efficacy of novel therapies for CHIKV arthritis. Chikungunya virus (CHIKV) is an alphavirus spread by mosquitos that was introduced to the Americas in 2013, causing two million infections across over thirty countries  and lossMouse models of CHIKV infection provide valuable information on the pathogenesis of arthritis ,8,9,10 aSpecifically, subcutaneous footpad inoculation of adult wild-type immunocompetent C57BL/6 mice with pathogenic strains of CHIKV results in acute foot swelling, myositis, tenosynovitis, and arthritis ,11,12,13This wild-type immunocompetent C57BL/6 mouse model has been used to evaluate responses to therapies during the acute CHIKV viremic period via drug administration at 3 days post-inoculation ,12,13. LThe housing of animals utilized in this study and the methods employed during animal experimental procedures were reviewed and approved by the George Washington University (GWU) Institutional Animal Care and Use Committee (IACUC), which complies with the guidelines stated in the National Institutes of Health’s (NIH) Guide for the Care and Use of Laboratory Animals. These experiments were conducted under the approved GWU IACUC protocol #A456.All studies with viable CHIKV were performed in certified BSL-3 laboratories in biological safety cabinets and were conducted under the approved GWU Institutional Biosafety Committee protocol #IBC-19-026.The chikungunya virus strain used in this study was an ECSA lineage, originally isolated from a human case in South Africa (SAH-2123), received from the World Reference Center for Emerging Viruses and Arboviruses . Our use of CHIKV strain SAH2123 from the East, Central, and South African (ECSA) lineage was based on the reported pathogenicity for this lineage as compared with others , i.e., tBriefly, following a single passage in Vero cells  in DMEM containing antibiotics and antimycotics, serial ten-fold dilutions of the virus stock were inoculated into six-well plates seeded, similarly, with Vero cells, as described previously , or werehttps://www.jax.org/ (accessed on 14 September 2021)) and inoculated at 11 weeks of age. Mice were housed in groups of five in micro-isolator cages in the ABSL3 laboratory at GWU. Mice were provided water and a standard commercial mouse food ad libitum throughout the experiments.C57BL/6J mice  were obtained from a distributor .Mice were then euthanized at various timepoints throughout the disease course . During experiments, animals were monitored a minimum of once daily for clinical signs associated with illness, including lethargy, abnormal posture, joint swelling, lameness, and limb gnawing. Mice were euthanized via COBlood (200 μL) was collected from the submandibular vein for serum analyses at 48 h post-inoculation to confirm infection and at 8, 10, 12, 14 and 16 DPI to measure viremia. Viral RNA was extracted using the Zymo Research Quick DNA/RNA Kit following the manufacturer’s protocol. Quantification of viral load was done with Invitrogen Superscript III Platinum One Step qRT-PCR Kit. RT-PCR (Invitrogen) was analyzed using a LightCycler 96 Instrument (Roche Diagnostics) with thermal cycling conditions as follows: one cycle at 50 °C for 900 s, one cycle of 95 °C for 120 s, and 45 cycles of 95 °C for 3 s, followed by 55 °C for 30 s and a cooling cycle of 37 °C for 30 s.Tarsal joint measurements were performed daily using calipers and were recorded, in mm, by animal identification number by day. Tarsal joint swelling was measured by daily measurement of the tarsal joint using calipers and averaging three measurements. The tarsal joint was measured directly posterior to the tarsal joint foot pad projection on the mouse foot, with only enough pressure to cause slightly flaring of the toes. The degree of inflammation was expressed as the increase relative to the pre-infection measurement (day 0), obtained by the following formula: [(x-day 0)/day 0)], where x is the footpad size measurement for a given day post infection.Immediately following euthanasia, hindlimbs were dissected with skin removed and placed directly in 10% formaldehyde for fixation for a minimum of 48 h at 4 °C.Bones were decalcified in an EDTA solution prepared by dissolving 41.4 g EDTA, 4.4 g sodium hydroxide, 50 g PVP in 1000 mL dH2O over very low heat with a magnetic stir bar until the solution was clear, taking up to 8 h. The solution was cooled to room temperature and the pH was adjusted to 7.0–7.4. After fixation, the tissue was washed in water, and placed in the EDTA solution at 4 °C with agitation changing the decalcification solution every other day to maintain pH between 7.0–7.4, for 28 days.After decalcification, the tissue was rinsed to remove salt before processing in 1× PBS three times for 5 min each, distilled water three times for 5 min each, 50% ethanol once for 5 min, and 70% ethanol once for 5 min, and stored in 70% ethanol at 4 °C until processing.Samples were then routinely processed for histopathologic evaluation. Briefly, samples were dehydrated in increasing concentrations of ethanol and embedded in paraffin wax. Subsequently, 5 μm-thick sections were adhered to positively charged glass slides. Sections were routinely stained with hematoxylin and eosin (H&E) for evaluation. Standard Masson’s trichrome stains were performed on select slides. All histologic evaluations were performed by a board-certified pathologist blinded to DPI and inoculation status. All slides were evaluated using an Olympus BK46 microscope and digital images were obtained using a Spot Imaging digital camera and Spot Pathsuite 2.0.A histologic inflammation scoring system was developed in order to have a semi-quantitative measure that can be used to compare outcomes of pathology in therapeutic studies. Based on a previous study , our scon = 8) demonstrated significant viremia 48 h post infection, with infectious titers ranging from 16 to 172 PFU/mL . At 25 DPI, the mouse with the inflamed extremity had synovitis with cartilage erosion, periostitis with mild cortical bone erosion and mild myositis with degenerative changes and a limited fasciitis.The uninoculated control mouse, who experienced low level viremia after being housed with infected mice, demonstrated mild inflammation with lymphocytic infiltrates in both feet. The other control mouse was free of any pathologic findings. In the infected mice, the inoculated feet also showed inflammatory changes, however. to a lesser degree than present in the locally infected extremity.Average histologic inflammation scores over time are shown in In all the infected mice, there was no evidence of granulomas, thrombosis or abscesses. Though articular cartilage and cortical bone erosions were present, there was no evidence of cartilage or bone necrosis, and no evidence of ischemic bone marrow necrosis or osteomyelitis. There was no evidence of soft tissue fat necrosis. There was no evidence of acute vasculitis nor fibrinoid necrosis. No cytopathic viral inclusions were identified.We present a wild-type immunocompetent C57BL/6 mouse model that can be used to study post-CHIKV arthritis therapies. This model differs from other models in that, in this model, histologic scoring is described to assess joint and muscle inflammation after the mouse is no longer viremic to mirror post-acute arthritis, as opposed to arthritis found during acute infection. Our key findings include that (1) all inoculated mice demonstrated viremia at 48 h post-infection, suggesting that a single 48-h post-inoculation blood draw for viremia assessment would be sufficient to confirm successful infection prior to investigational drug therapy as part of an experimental protocol; (2) there is a window, from 16–21 DPI, wherein the mice are no longer viremic but still display prominent histologic evidence of inflammation, that would be the ideal window for studies of investigational drugs; (3) the composite histologic inflammation score may be of utility for determining efficacy outcomes; and (4) tarsal joint swelling is only present for a limited duration during viremia, which excludes tarsal joint swelling as a valuable endpoint for post-acute arthritis of chikungunya drug studies.Our findings are similar to the findings of other studies, including the early presence of viremia in adult immunocompetent mice after inoculation with multiple different strains of CHIKV ,9,10,20 Our histopathologic findings differ from Goupil et al. in that we did not find the other features of necrosis described in that report, including necrosis of cartilage or bone, nor evidence of ischemic bone marrow necrosis, and we saw no evidence of osteomyelitis. Furthermore, we did not identify bone marrow necrosis of an ischemic etiology or pathogenesis, nor any local or regional vascular pathology that would contribute to vascular compromise. Moreover, the marrow necrosis of an ischemic pathogenesis should demonstrate fat necrosis with multinucleated giant cell response that was not identified in our histologic images. Finally, the neutrophilic infiltrates found in Goupil et al.’s photomicrographs, which may be interpreted as active osteomyelitis, were also not present in our cases.This is the first study to our knowledge to follow the mice to 25 DPI. We saw resolution of histologic inflammation in some mice at 25 days, indicating that endpoints for drug studies should be assessed prior to 25 DPI.We also found that tarsal joint swelling was only significant for a duration of approximately 10 days during viremia, which may preclude the use of tarsal joint swelling as a valuable endpoint for post-CHIKV arthritis drug studies. Another study by Lee et al.  documentA limitation of this study includes the limited number of mice utilized; however, the similarity of our findings in terms of severity and duration of disease with those in other studies is reassuring. Another limitation was the use of a histologic inflammation score that is semi-quantitative as opposed to quantitative. A quantitative score could be developed by counting the numbers of inflammatory T-cells identified by immunohistochemistry per high-power field. However, the benefit of the semi-quantitative score used in this study is that it generates a composite score from multiple elements of tissue histology that contribute to the inflammatory pathology. The composite score is based on a specific assessment of the joint pathology resulting from CHIKV infection and so may be especially well suited to represent clinical response to disease/therapy. In addition, future validation of this model, with arthritis therapies of various durations of treatment, is needed.We presented a wild-type immunocompetent C57BL/6 mouse model for studying post-acute arthritis of chikungunya. Our findings suggest the optimal timeframe for testing novel arthritis therapeutics is at 16–21 DPI, using histologic outcomes. Validation of this model using various arthritis therapies is needed. This may serve as a foundation for future studies to develop mouse models for studying chronic post-infection arthritis in other alphaviral infections known to cause arthritis, as well.


Enterococcus faecalis and Enterococcus hirae could be identified at the species level with an accuracy of 96.4% to 97.5%. We also demonstrated that the absolute counts of various bacteria could be determined by spiking the sample with a bacterium as an internal standard. Then, we tested whether this convenient bacterial identification method could evaluate the antibiotic sensitivities of multiple bacteria simultaneously. In order to evaluate antimicrobial susceptibility, a mock community, an artificial mixture of the nine bacterial strains, was prepared and cultured in the presence of the antibiotics ofloxacin or chloramphenicol, and the 16S rRNAs were analyzed by using Nanopore sequencer. We confirmed that antibiotic-induced cell count reductions could be measured simultaneously by quantifying the abundances of various bacteria in the mock community before and after culture. It was thus shown that the antibiotic sensitivities of multiple bacteria could be evaluated simultaneously, with distinction made between bactericidal action and bacteriostatic action. This methodology would allow rapid evaluation of antibiotic activity spectrum at the species level containing a wide variety of bacteria, such as biofilm bacteria and gut microbiota.While amplicon sequencing of 16S rRNA is a common method for studying microbial community, it has been difficult to identify genera and species using next-generation sequencers to examine some regions  because of the short read lengths. However, the advent of third-generation sequencers has made it possible to analyze the full length of the 16S rRNA gene, which allowed for species level identification at low cost. In this study, we evaluated the accuracy of the identification with a third-generation sequencer, MinION from Oxford Nanopore Technologies, using nine indigenous bacteria that can pose problems with food poisoning and opportunistic infections as an example. We demonstrated that  Bacteria exist in complex communities in all environments, bringing about various human health issues, including food contamination , tooth dRecent advances in sequencing technology have made it possible to comprehensively analyze bacterial taxonomy and abundance in large diverse bacterial populations. At present, sequencing a particular region of the 16S rRNA gene using a next-generation sequencer (NGS) is the most commonly used method for microbial community analysis . It has In this study, we tested whether measuring bacterial abundance by sequencing instead of colony counting could be applied to antibiotic susceptibility test . The absKocuria rhizophila (ATCC 9341), Escherichia coli (ATCC 33588), Enterococcus hirae (ATCC 8043), Bacillus subtilis (ATCC 6051), Enterococcus faecalis (ATCC 47077), Streptococcus mutans (ATCC 25175), Listeria innocua (ATCC33090), and Staphylococcus epidermidis (ATCC12228). Ralstonia solanacearum (T-11 strain) [Lactobacillus sakei (NBRC 15893) used for internal standard was obtained from the National Institute of Technology and Evaluation (NBRC). All these strains were cultured in a BD Difco Nutrient broth supplemented with 5 g/L yeast extract (NYB).Nine test strains and one internal standard strain were used. The following test strains were obtained from the American Type Culture Collection (ATCC);  strain)  was isol660nm of 0.005. An antibiotic chloramphenicol  or ofloxacin  was added to the bacterial suspension to achieve a final concentration of 0.25–32 µg/mL, and the suspension was incubated at 30°C in standing culture for 24 hours. The OD660nm was measured using a plate reader. After culture, the minimum concentration at which the OD660nm did not exceed 0.02 was taken as the minimum inhibitory concentration [Thawed cells from 20% glycerol stocks of the test strain was diluted in NYB, cultured 18 hours at 30°C and diluted in a fresh medium to an optical density (OD)ntration .5’- AGAGTTTGATCCTGGCTCAG-3’, 1492R primer [5’-GGTTACCTTGTTACGACTT-3’) and KAPA HiFi HotStart ReadyMix . The PCR thermal profile consisted of an initial denaturation of 4 min at 95°C, followed by 30 cycles of 20 s at 98°C, 15 s at 60°C, 30 s at 72°C, and a final step of 2 min at 72°C. The amplification product was purified using the FastGene Gel/PCR Extraction Kit  according to manufacturer’s protocol and the concentration was determined using a Qubit ® Fluorometer . Nanopore sequencing samples were prepared to check the taxonomy identification performance  according to manufacturer’s protocol. Polymerase chain reaction (PCR) was performed using primers that bind to both ends of the 16S rRNA gene  by the copy number of the 16S rRNA gene present in each strain. CFUs were counted as below. The test strains were incubated for 18 hours at 30°C in NYB medium, each strain was collected by centrifugation , washed with phosphate-buffered saline, serially diluted in phosphate-buffered saline, and 20 µL was spotted 3 times onto agar plates prepared with ATCC recommended medium of each strain. Plates were incubated for 24 hours at 30°C and CFUs were measured (20–100 colonies were counted for each strain). The antibiotic chloramphenicol or ofloxacin was added to the mixed culture, and the strain was incubated in standing culture at 30°C for 6 hours. After incubation, a known bacterium was added as the internal standard for absolute quantitation. The internal standard consisted of 25 µL of a suspension of Lactobacillus sakei at an OD660nm of 2.0. The use of a taxonomically distant strain as the internal standard facilitates the identification of the reads from the internal standards. We selected L. sakei because it differs at least at the family level from each of the test strains. The DNA was then extracted using the ZymoBIOMICS DNA Miniprep Kit to prepare a library following the manufacturer’s protocol.The test strain that cultured 18 hours at 30°C was centrifuged, the resulting supernatant was removed, cells were resuspended in fresh medium and the suspension of all strains were mixed to contain equal amounts of the rRNA gene at an ODThe 16S rRNA amplicon Library preparation was performed using the 16S Barcoding Kit 1–24  according to the manufacturer’s protocol. The PCRs were conducted using a primer contained in the kit and LongAmp Taq 2X Master Mix. After purifying the PCR product with Agencourt AMPure XP beads , the amount of eluted DNA was determined using Qubit® Fluorometer. PCR amplicon (100 ng of DNA in 10 µL buffer) was added to 1 µL of rapid adapter (Oxford Nanopore Technologies) and incubated at room temperature according to the manufacturer’s protocol of Rapid Sequencing Kit (Oxford Nanopore Technologies). Sequencing solution was prepared by mixing amplicon library (11 µL), sequencing buffer (34 µL), loading beads (25.5 µL) and water (4.5 µL), and loaded on MinION R9.4.1 SpotON Flow Cells. The sequencer was controlled using the MinKNOW software v3.6.5. For each sample, more than 120,000 reads having a Q-score exceeding 7 were acquired and used for the analysis. Fast5 files were base-called, de-multiplexed and trimmed barcode and adapter sequences using guppy v3.2.10 (Oxford Nanopore Technologies). The base-called sequences (FASTQ files) were mapped to the database of bacterial 16S rRNA sequences from NCBI RefSeq Targeted Loci Project using Minimap2 . Genus nEscherichia also revealed high accuracies of 98% to 99%, which is nearly the accuracy at the family level. For Escherichia, 18.4% of reads were classified as the Shigella genus in the same Enterobacteriaceae family. The accuracy of identification at the species level varied depending on the strain, and E. hirae and E. faecalis could be identified at high accuracy of 96.4% and 97.5%, respectively. In the following analyses, E. coli were classified at the family level, E. hirae and E. faecalis at the species level, and other bacteria at the genus level. Next, to determine the accuracy of the abundance ratio measurement, a mixture containing 16S rRNA genes from nine strains in a known ratio were sequenced. When the abundance of each strain was 0.2–50% , and brain heart infusion broth (BHI). When nine test strains were cultured separately in each medium, some test strains proliferated explosively while others slowly, indicating that there were several orders of growth rate differences. Because it was likely that, when mixed, fast-proliferating strains would consume all nutrients and others die without the use of antibiotics, we searched for media that would allow all strains to proliferate at similar rates. When cultured in a NB supplemented with yeast extract (NYB) for 6 hours, each test strain increased by 4–16 fold . Since tTo determine the concentration of antibiotics to use in the mixed culture, each test strain was cultured separately and sensitivities to the antibiotics ofloxacin and chloramphenicol were measured . The tesRalstonia, Bacillus and Enterobacteriaceae, which are highly sensitive to ofloxacin . When chloramphenicol was added  alone, which is an obstacle to achieving the depth of identification. Some studies suggest full length of the 16S rRNA gene is needed for bacterial identification at the genus and species levels ,25. AnalShigella , could bStreptococcus or Staphylococcus at the species level [The disadvantages of the Nanopore sequencer compared to NGS is higher misclassification rates. Although previous studies suggested that the Nanopore sequencing could identify some bacteria of the genus es level –16, it wes level ,27, are The 16S barcoding kit includes 24 barcode sequences, enabling an examination of 24 conditions × 9 bacterial species (216 reactions) in a single experiment. In this study, we acquired 120,000 reads per sample by 18 hours sequencing, whereas Mitsuhashi et al.  reportedAs we were able to quantify purified rRNA gene amplification products at the species level by using Nanopore sequencing and our analytical method, we then tested whether the absolute amounts of genes could be quantified in a medium containing multiple bacterial strains. This technique requires three conditions. First, the difference in DNA extraction efficiency among different bacteria must be minimized. This condition could be satisfied using the ZymoBIOMICS DNA Miniprep Kit, which has been shown to be an effective extraction method for 16S rRNA amplicon analysis referred in several papers ,29.Second, the absolute amounts must be quantified, which could be achieved with an internal standard of bacterial cells added at known concentrations. Such a method for microbial community analysis had been used with NGS , but notS. mutans did not proliferate in NB at all, the proliferation rates in other media suggested that this strain could proliferate in media containing yeast extract. Therefore, we prepared NB supplemented with yeast extract (NYB), and confirmed that all of the test strains grew equally in this medium. We considered that bactericidal action and bacteriostatic action could be distinguished by using the medium.Third, it is necessary to select a medium that allows concurrent growth of multiple bacterial species because the appropriate nutrient medium for growth differs among bacterial species. To find an appropriate medium, we cultured all test strains using Luria–Bertani medium, MRS broth, tryptic soy broth, NB, and brain heart infusion broth recommended for the respective strains, and found that NB produced smallest differences in proliferation rate among the strains. Although Ralstonia, Bacillus, and Enterobacteriaceae, decreased markedly, whereas that of the other strains increased slightly. This result suggested that the antibiotic had little impact on the six surviving strains, and that their proliferation rates increased as a result of the availability of nutrients that would have been consumed by the three strains killed by the antibiotic. When the ofloxacin concentration was increased, none of the strains proliferated, and three strains of Ralstonia, Bacillus, and Enterobacteriaceae, decreased to 4% of the initial amount. This result, combined with the fact that ofloxacin is an antibiotic that kills bacteria by inhibiting their DNA gyrase [Kocuria. Chloramphenicol is known as a bacteriostatic agent that binds to ribosomes to stop protein synthesis [Kocuria decreased from the initial count. This bacterium might have died gradually under stress from other bacteria when its proliferation was inhibited by chloramphenicol. For the test strains in this study, the bacterial count reduction due to antibiotics differed 10 fold between the bactericidal antibiotic (ofloxacin) and the bacteriostatic antibiotic (chloramphenicol). However, to apply the present technique to other bacteria or novel antibiotic candidates, it is necessary to clarify the boundary statistically between bactericidal and bacteriostatic antimicrobials using known antibiotics. Moreover, caution should be exercised to prevent the occurrence of bacterial death by bacteriocin [We showed that the antibiotic sensitivities of nine bacterial strains in mixed culture could be measured using a Nanopore sequencer. When ofloxacin was added at a concentration of 0.25 µg/mL, only the level of highly sensitive bacteria, i.e., A gyrase , suggestynthesis , and theteriocin  or the tteriocin  due to sIn conclusion, this study demonstrated the utility of Nanopore sequencing of 16S rRNA amplicons as an approach to evaluate the antimicrobial susceptibility by analyzing the absolute abundance of test species. Although conventional method by 16S rRNA amplicon sequence can only analyze the relative amounts of bacteria, the absolute abundances of all species in a bacterial mixture can be measured simultaneously just by spiking the sample with an internal standard strain. Our method is quicker than colony counting, and is applicable to an antimicrobial susceptibility testing that can distinguish between bactericidal and bacteriostatic effects. Further improvements in the mixed culture conditions to ensure survival of the test species will allow for application of the method to various bacteria, such as biofilm bacteria and gut microbiota.S1 Fig(TIF)Click here for additional data file.S2 FigThe test strains mixture was incubated 6 hours. The absolute abundance of each test strain was calculated by comparing sequencing reads of test strain and internal standard strain. The abundances fold change of after incubation relative to before incubation were shown in Y axis.(TIF)Click here for additional data file.


Within Calophyllaceae we observed a longer LSC and reduced IRs in Mahurea exstipulata, resulting in some genic rearrangement, and a short inversion (psbJ–psbE) in Kielmeyera coriacea. Phylogenetic analyses recovered the clusioids and the five families as monophyletic and revealed that conflicts in relationships reported in the literature for the group agree with nodes concentrating uninformative or conflicting gene trees. Our study brings new insights about clusioid plastome architecture and its evolution.A complete chloroplast genome is not yet available for numerous species of plants. Among the groups that lack plastome information is the clusioid clade , which includes five families: Bonnetiaceae, Calophyllaceae, Clusiaceae, Hypericaceae, and Podostemaceae. With around 2200 species, it has few published plastomes and most of them are from Podostemaceae. Here we assembled and compared six plastomes from members of the clusioids: five from Calophyllaceae (newly sequenced) and one from Clusiaceae. Putative regions for evolutionary studies were identified and the newly assembled chloroplasts were analyzed with other available chloroplasts for the group, focusing on Calophyllaceae. Our results mostly agree with recent studies which found a general conserved structure, except for the two Podostemaceae species that have a large inversion ( This is due to their high number of copies in a cell , their much smaller size when compared to nuclear genomes (ca. 120 genes8vs. thousands of genes9), and the availability of numerous tools to assemble chloroplast genomes . Thus, it usually is easier to obtain the DNA from the chloroplast when compared to the nucleus. In addition, sequencing the chloroplast is cost advantageous, and the generated data require less computational power to be processed and analyzed. Despite the existence of a canonical chloroplast structure for land plants14, which includes two unique regions , and one duplicated region (the inverted repeat-IR), much variation has been already found at different taxonomic levels6. Finally, most of the chloroplast genes are functional, thus usually conserved15. These reasons make plastid DNA extremely attractive for high throughput sequencing. Furthermore, the possibility of using the chloroplast genome from a phylogenetically distant taxon as a reference for the assembly accelerates data processing. However, it is important to mention that the analysis of nuclear genes is becoming much easier16.Since the advance of next-generation sequencing complete plastomes or chloroplast coding sequences have been the most used source of information to explore phylogenetic relationships among plants at diverse scales17, there is evidence that these circular molecules represent only a small fraction of the total chloroplast DNA18. Indeed, chloroplast chromosomes are hypothesized to consist of branched and complex multigenomic forms19. Even though phylogenetic conflict within plastid data has been known for over two decades21, it was largely neglected until recently, when the traditional approach of treating the plastome as a single “supergene” has been questioned23, creating some debate (see Doyle 2021)24. There is a growing body of evidence showing that plastid regions contribute differentially to phylogenetic support27 and that several of the plastome genes actually are uninformative26. Finally, heteroplasmy was recently quantified and may be more common than previously expected28. This phenomenon allows the occurrence of heteroplasmic recombination, which is a potential source of gene tree conflict25. Besides heteroplasmic recombination, heterotachy, stochastic and systematic errors and horizontal gene transfer have been invoked as potential causes of gene tree conflict. These findings are already changing how we see plastomes and will probably influence future works using plastid data.Although the chloroplast genome is generally imagined as being a quadripartite circular structure32. Intrafamilial relationships remained poorly understood until Xi and collaborators (2012)33 presented a phylogeny based on 82 plastid, six mitochondrial, and three nuclear genes and suggested relationships between families in the order, albeit some were only poorly supported. Indeed, recently Cai and collaborators (2021)34 described what they called “the perfect storm”—a combination of incomplete lineage sorting, introgression, and gene tree estimation error that may occur simultaneously during periods of rapid radiation, making phylogenetic inference challenging—and showed the difficulties in resolving relationships especially along the backbone of Malpighiales. Within the order, the clusioid clade includes five families , 94 genera and ca. 2200 species36. Relationships within the clusioids had remained unclear—indeed, the very existence of the group was questionable—until somewhat over a decade ago. For example, Wawra (1886)37 included some of the taxa in Ternstroemiaceae, today included in the Theaceae , and Cronquist (1981)38 separated them in different orders and subclasses, while where Podostemaceae were to be placed was almost literally anybody’s guess. On the other hand, the families Hypericaceae and Calophyllaceae have frequently been included in Clusiaceae s.l. .The highly diverse order Malpighiales includes 36 recognized families in its most recent circumscription and represents ca. 7.8% of eudicot diversity36. The authors recognized two major clades: (Clusiaceae + Bonnetiaceae) and ). Xi and collaborators (2012)33 recovered the five clusioid families as monophyletic and in a well-supported clade and the same topology from Ruhfel et al. 36; the clade Bonnetiaceae + Clusiaceae had the lowest support (85% bootstrap/0.92 Bayesian posterior probability). Recently, Cai and collaborators (2021)34 also recovered the clusioid families in a well-supported clade using 423 single-copy nuclear loci, but internal relationships were very different—))—although poorly supported; Podostemaceae were not included. The conflicting relationships within the clusioids may result from the above-mentioned factors, i.e., “the perfect storm”, but also indicate that nuclear and plastid data have conflicting evolutionary stories, since most of Xi et al.33 data came from plastid genes. The use of chloroplast genomic data to investigate intergeneric relationships and/or genome structure within the clusioids is recent. The studies have focused mainly on Clusiaceae6 and Podostemaceae39. Jin and collaborators (2020)40 included Calophyllaceae, but only one plastome, i.e., that of Mesua ferrea L.The relationships between the families included in the clusioid clade were explored in more depth by Ruhfel and collaborators 31. Within the Calophyllaceae two tribes are currently recognized: Endodesmiae, including two monotypic African genera (Endodesmia Benth. and Lebrunia Staner), and Calophylleae, including the remaining 12 genera , which are distributed throughout the tropics. In both Ruhfel et al.41 and Cabral et al.42Endodesmia was well-supported as sister to the remaining genera of the family (Lebrunia was not included in either study). Relationships among the genera in Calophylleae are not completely understood, e.g., the position of Calophyllum + Mesua clade may be (1) sister to the clade Kayea + Poeciloneuron43, (2) sister to a clade that includes Mammea, Kayea and Poeciloneuron35, or (3) sister to a clade including the neotropical genera Caraipa, Clusiella, Haploclathra, Kielmeyera, Mahurea, and Marila42.The family Calophyllaceae is pantropical and includes 14 genera and ca. 460 speciesCalophyllum, Caraipa, Kielmeyera and Mahurea), represented by the species Calophyllum brasiliense Cambess., Caraipa heterocarpa Ducke, Kielmeyera appariciana Saddi, K. coriacea Mart. & Zucc., and Mahurea exstipulata Benth. We also assembled and annotated the first plastid genome from Clusia L., the largest genus in the Clusiaceae. Our data were analyzed in the context of the clusioid clade, and we hope to improve our understanding of the evolution of the group.Complete plastome information, including its annotation, is still not available for many species. Thus, the present work aims to reduce this knowledge gap for the family Calophyllaceae by presenting four complete and one nearly complete newly assembled and annotated plastid genomes from four different genera in the family  and the smallest IRs , respectively 9,923 to 12,143 bp longer and 8,966 to 10,781 bp shorter respectively than those of the other species. All species presented an SSC similar in size ranging from 17,464 bp in C. brasiliense to 19,102 bp in Clusia panapanari (Aubl.) Choisy. The GC content was similar among all species , and four rRNAs  Spreng.) being the most distinct.Five plastomes have 87 protein-coding genes, 37 transfer RNA (tRNA) and eight ribosomal (rRNA), totaling 132 genes. However, rpl22 on the LSC side and by rpl2 on the IRb side, with rps19 spanning the junction in all species but M. exstipulata. This species lacks rps19 in this region and has ycf2 flanking the junction on the LSC side and trnL on the IRb side. The IRb-SSC junction always has the ycf1-fragment on IRb and the ndhF on SSC, while ycf1 spans the SSC-IRa junction. Finally, the IRa-LSC junction is flanked by rpl2 and rps19 on the IRa side and by trnH on LSC for all species, although trnL is on the IRa side in M. exstipulata.Few differences in the limits of the four regions of the plastomes were observed Fig. . The LSCtrnK-UUU–rbcL inversion in both Podostemaceae species. Another inversion (psbJ–psbE) was observed only in K. coriacea. There were no other major structural differences between the assessed plastomes  Blume most of them were forward and palindromic repeats; complementary repeats were found only in Garcinia gummi-gutta (L.) N.Robson, which has two of them , and in some introns  , and hexanucleotide SSRs were found only in five species  and that eight of them are CDSs. Nine of the ten regions with more segregating sites and more estimated mutations were the same; at least eight of them are CDSs, and six of them are in the LSC. All the ten regions with highest nucleotide diversity were intergenic spacers and seven out of ten are in the LSC. Overall, LSC and SSC had higher variability  when compared to the IRs (Supplementary Table 50 (nogb) has 77,015 bp, including 34,595 distinct patterns, 21,558 parsimony-informative sites, and 21.16% of gaps/missing data. The alignment processed on Gblocks50 (gb) has 66,671 bp, including 30,582 distinct patterns, 19,548 parsimony-informative, and 12.94% gaps/missing data. We used three different methods  and eight different datasets  in our analyses.Our 59 sequences complete alignment prior to the removal of poorly aligned regions on GblocksKielmeyera species grouped in a clade, Caraipa heterocarpa being sister to this clade; Mahurea exstipulata was recovered sister to Kielmeyera + Caraipa clade, and Mesua ferrea and Calophyllum formed another clade. The position of Mammea americana was not stable and will be discussed below. Within Podostemaceae, Tristicha trifaria was recovered sister to the clade M. foeniculaceum + Podostemum ceratophyllum. In Hypericaceae, Cratoxylum cochinchinense was recovered sister to the clade Vismia ferruginea + Hypericum; relationships within Hypericum were not stable. In Clusiaceae, the two Clusia species included in the study grouped in a clade which was recovered sister to the clade including the two Garcina species. The two Bonnetiaceae genera, represented by B. paniculata and Ploiarium sp., were recovered in a clade. All stable branches mentioned above received 100% (ultrafast bootstrap—UB)/1 (posterior probability—PP) support values.The trees we obtained exhibited the same topology for most relationships. In all analyses the clusioids were recovered in a strongly supported monophyletic clade with all the five families also being monophyletic. Calophyllaceae was recovered sister to a clade composed by Podostemaceae and Hypericaceae. The position of Bonnetiaceae and Clusiaceae changed in different analyses and datasets. Within Calophyllaceae most relationships were stable with the two Mammea , and (3) the relationship between Hypericum fraseri Steud. and H. kalmianum L. (Hypericaceae). These relationships are represented in the network . PhyParts51 gene tree discordance analyses using MC trees after removal of poorly aligned regions  as input showed that the two of three branches with lower support in our analyses are the ones where there is more gene conflict  the position of the Bonnetiaceae and Clusiaceae, (2) the positions of ork Fig. . Conflicork Fig. -IIA. Onlork Fig. -IIB. Supeae Fig. -IIC. Alleae Fig. -IID. In Calophyllum brasiliense) presented here showed a conserved structure with total size, general organization in a quadripartite structure, IR, LSC and SSC sizes, number of genes and GC content in agreement with the values for an ordinary angiosperm plastome17. However, as is the case in several groups, our study focusing on Calophyllaceae revealed that there is variation of plastome structure for the family. Mahurea exstipulata chloroplast had only a single copy of the usually duplicated genes rpl2, rpl23 and ycf2, which moved from the IRs to the LSC, resulting in the contraction of former and the expansion of the latter and in distinct IR/single-copy regions junctions. The expansion or contraction of IRs is a common type of rearrangement in plastomes that was already documented for the clusioids in Podostemaceae40 and for some other groups like Geraniaceae52, Acanthaceae53 and the genus Passiflora46. However, this phenomenon was not observed in Calophyllaceae until the present study. Although Podostemaceae species and M. exstipulata share IR contractions, their plastomes are quite distinct and these contractions represent independent evolutionary events. In M. exstipulata the IR contraction resulted from the loss of one copy of the genes rpl2, rpl23 and ycf2 and it was associated with a large expansion of the LSC; it is between 9,258 and 19,040 bp longer when compared to the other clusioids. The loss of a copy of the same three genes is a rare event and has been reported only in Strobilanthes cusia (Nees) Kuntze 53, and the loss of rpl2 and rpl23 is known from Cuscuta reflexa Roxb. 54 and Lonicera japonica Thunb. 55 On the other hand, in Podostemaceae the IR contraction resulted from both gene (ycf1 and ycf2) and intron (rps16) loss and pseudogenization of accD, rpl22, and clpP, all associated with a reduction also in the single copy regions, which resulted in a smaller chloroplast as a whole. Among the newly sequenced plastomes, all species but M. exstipulata had the junctions between IR and LSC and SSC regions similar to those of a canonical angiosperm plastome56. The differences observed in the limits of the four main parts of the plastome correspond to one of the sources of variation in the plastome structure and have been described from different taxonomic levels, e.g., within Calophyllaceae and between the clusioids or within Cercidoideae legumes56. The usually slower evolutionary rates of genes IRs when compared to single copy regions is widely reported in the literature57 and was also confirmed in our study. Regarding genes and intergenic spacers, the latter were the most variable and should be considered in future evolutionary studies. In agreement with Walker and collaborators (2019)25, the rpoC2 was the gene with more segregating sites.Five out of the six newly assembled plastomes 39 and Jin et al. (2020)40. Although Jin et al. (2020)40 also reported this inversion for Cratoxylum cochinchinense (Hypericaceae) and suggested it could be a synapomorphy of the clade composed by these two families, it was observed only in Podostemaceae in our study. We also registered the first inversion within Calophyllaceae (psbJ-psbE) in Kielmeyera coriacea. Repetitive regions are known to flank inversion breakpoints. In our data, it was observed only in Podostemaceae 58 the loss of intron 1 from that gene in tobacco resulted in phenotypically mutant plants and prevented the splicing of intron 2.Regarding other structural differences noted in the clusioid chloroplasts, the et al. 20939 and J59, Chase et al. (2002)60 and Gustafsson et al. (2002)61 all used rbcL to infer relationships and recovered Podostemaceae (P) sister to Hypericaceae (H) and Calophyllaceae (Ca) sister to Clusiaceae s.s. (Cl) with Bonnetiaceae (B) as sister to the Podostemaceae + Hypericaceae clade 60 or as sister to a clade including all the other four families 61. In all three studies, support values for the position of Bonnetiaceae and for Clusiaceae + Calophyllaceae were moderate to low and the monophyly of the clusioids was poorly supported. A different topology was proposed by Wurdack and Davis (2009)62 who analyzed 13 genes from all three compartments and recovered ((Cl + B) + (Ca (H + P))) and confirmed clusioid monophyly. Similarly, Soltis et al. (2011)63 recovered different relationships using 17 genes, most of them from the chloroplast: (((P + H) + Ca) + B) + Cl)). In the same year, Ruhfel et al. (2011)35 using three plastid and one mitochondrial genes and the most extensive sampling of the clade so far (194 species included), recovered the same topology of Wurdack and Davis (2009)62 as did Xi et al. (2012)33 using genes from the three genomic compartments and Jin et al. (2020)40 using 82 protein-coding plastid genes. More recently, Cai et al. (2021)34 recovered a distinct topology using a 423 nuclear gene matrix: (((Ca + B) + H) + Cl)-Podostemaceae were not included, however, there is much incongruence between gene trees and the species tree. Our results mostly agree with the topology from Wurdack and Davis (2009)62, but also reveal much gene tree disagreement, particularly for two relationships: (1) Clusiaceae + Bonnetiaceae that is supported by only 340 of the total 2050 gene trees and 18 of the total 82 gene trees, and (2) Calophyllaceae + (Hypericaceae + Podostemaceae) is supported by 500 of the total 2050 gene trees and 21 of the total 82 gene trees 30, Clusia and Garcinia L. being the only genera with more than one representative and recovered as monophyletic in all our analyses. Within Hypericaceae, our results recovered the same relationship as found by Ruhfel et al. (2016)41, with Cratoxylum cochinchinense (Cratoxyleae) + (Vismia ferruginea Kunth. (Vismieae) + Hypericum spp. (Hypericeae)). Within Calophyllaceae, only representatives from the tribe Calophylleae were included in the present study. Ruhfel et al. 41, recovered Mammea in a clade with Kayea and Poeciloneuron, and this clade was sister to a clade including Calophyllum and Mesua. Recently, Cabral et al. (2021)42 evaluated relationships between Calophylleae genera. The authors recovered Mammea in a clade with Poeciloneuron alone and this clade as sister to a large clade including the genera Calophyllum, Caraipa, Clusiella, Haploclathra, Kielmeyera, Marila, Mahurea, and Mesua, although with low support. Our analyses recovered both positions for Mammea. Mammea was recovered as sister to the clade Calophyllum + Mesua, as in Ruhfel et al. 41, in all four coalescent schema, i.e., 82gb, 82nogb, 2050gb and 2050nogb, but all with low support  42, with high support (100% UB/1.0 PP), and the relationship between the clade  and the clade with the remaining genera was moderately to strongly supported  . However, despite scarce, there is evidence of recombination in chloroplasts , raising a question that deserves further exploration. Thus, it would be interesting to see studies testing for non-recombinant units in the plastid prior to phylogenetic inference. There is a lot to be learned about organellar genomes, including a deeper investigation of its multibranched structure.The conflicts reported here show that partitioning the dataset, filtering for poorly aligned regions and the method of inference can impact the topology of the tree. The use of MSC, considering each plastid gene individually, was recently recommended to infer phylogenies with plastid data Clusia and Calophyllum, with around 300–400 and 200 species, respectively. Whenever possible, it should be combined with data from other genomic compartments. Obtaining genomic DNA from herbarium specimens to target nuclear regions is still challenging, although it may become more common with the development of probe sets that can be used in different groups and of standardized protocols16. In this scenario, chloroplast DNA data availability will also increase since this information is being recovered with nuclear DNA in target enrichment capture due to the presence of plastids in high copy number in a cell. Furthermore, an initial exploration may help to point out groups where more sampling is needed or where relationships will need a different source of data to be clarified.Plastid data can be a good starting point to investigate phylogenetic relationships, particularly in large and neglected genera such as34 have shown with nuclear data is that reconstructing phylogenies in Malpighiales, to which the clusioids belong, represents a huge challenge due to incomplete lineage sorting, tree error estimation and gene flow. Therefore, for a better comprehension of phylogenetic relationships, more analytical refinement is required, such as testing different partitioned schema.The disagreements found in this study have been reported since the first molecular phylogenies for the clusioids appeared. We now need to add data from other genomic compartments . However, what Cai et al. (2021)Kielmeyera appariciana, K. coriacea, Caraipa heterocarpa and Mahurea exstipulata were sampled during the development of R. J. Trad and F. N. Cabral theses and vouchers have been deposited in the University of Campinas herbarium (UEC). All the plants included in the study were collected with ICMBio fieldwork permits (n. 23954–4 and 41896–3 to R.J.T. and n. 33308 to F.N.C.), and INPA permit to collect in the Ducke Reserve for F.N.C. Collections and experimental research on the plants present in the study complied with international, national, and/or institutional guidelines. Kielmeyera specimens (Trad 192—K. appariciana and Trad 401—K. coriacea) were identified by Rafaela Jorge Trad and Caraipa and Mahurea specimens  were identified by Fernanda Nunes Cabral. A sample of Calophyllum brasiliense was obtained from the specimen N. Hind 4260 from UEC herbarium; its identification was confirmed by Maria do Carmo Estanislau do Amaral and Volker Bittrich. Reads of Clusia panapanari were downloaded from GenBank (accession number SRR7518735). Complete voucher information is presented in Supplementary Table Silica dried leaves of 67 modified by Caddah68. The DNA was quantified on NanoDrop Spectrophotometer  or on Qubit 2.0 . Genomic libraries for whole-genome were prepared and sequenced 2 × 150 bp on Illumina NextSeq 500 Mid-Output by Genohub . All adapters were removed by the company.Total DNA of six samples was extracted following Doyle & Doyle11 with the following parameters: -R 15 -k 21,35,45,55,65,75 –max-reads 4E7 -F embplant_pt. All assembly graphs from GetOrganelle11 were visually checked in Bandage v 0.8.169, so parameters could be adjusted. For all species two configurations of the plastome were assembled. For Calophyllum brasiliense and for Kielmeyera coriacea, plastomes were assembled in a different way. (2) Reads were mapped to the closest chloroplast available at the time this study started, i.e., Garcinia mangostana L. (GenBank accession NC_036341.1) with Bowtie2 v 2.2.170 plugin on Geneious 912 by adding the command –no-discordant to the default parameters; mapped reads were assembled with Platanus v.1.2.471. Since the two haplotypes found are present in the same proportion in a cell72, we chose the same configuration of the plastome published for G. mangostana to use in subsequent analysis. To validate our assemblies and to verify the coverage, reads were mapped to the assembled genomes using Bowtie2 v 2.2.170 as mentioned above but using the assembled plastome as reference. Between 49,148 and 540,562 reads mapped the respective assembled chloroplast (Table Reads were assembled in two ways. 1) Using GetOrganelle v.1.6.4 Using Ge73 implemented on Chlorobox website (https://chlorobox.mpimp-golm.mpg.de/). The annotation was checked and corrected on Geneious 912. Start and stop codons were checked for all the genes and tRNAs limits were checked using ARAGORN74 output from Chlorobox (https://chlorobox.mpimp-golm.mpg.de/). Potential pseudogenes were defined by Blast following Silva et al. (2018)75. Finally, circular gene maps were generated with OGDRAW76. All subsequent analyses, except IRscope77, were performed on plastomes with only one IR to avoid redundant results.Assembled chloroplasts were automatically annotated using GeSeqBonnetia paniculata Spruce ex. Benth. (Bonnetiaceae), Mesua ferrea L.  Garcinia gummi-gutta (Clusiaceae), Cratoxylum cochinchinense (Hypericaceae), Marathrum foeniculaceum (Podostemaceae) and Tristicha trifaria (Podostemaceae). All the GenBank accession numbers are found in Table 12. Beginning in the same region is important to avoid Mauve infer false rearrangements. A total of 12 plastomes were aligned using progressive Mauve algorithm in Mauve Plugin v. 2.3.2 in Geneious 912 to check for structural differences such as inversions or rearrangements. IR boundaries were evaluated on IRScope online77.The gene content was compared between the five Calophyllaceae and the Clusiaceae newly assembled plastomes Table . For gen17, REPuter44 was used to identify direct, complement, palindromic, and reverse repeats with the following parameters: minimal size of 30 pb and Hamming distance of 3. For Calophyllum brasiliense and for Kielmeyera coriacea the Ns and IUPAC ambiguous bases had to be manually removed from the respective fasta file in order to run REPuter44. And MISA49 was used to identify simple sequence repeats (SSR) with a minimum number of 7, 4, 4, 3, 3, and 3, for mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. The parameters followed Silva et al. (2019)78.Since reorganizations can be associated with small dispersed repeats79 or on Geneious 912 and aligned on MAFFT v. 7.30880 with the ‘adjustdirection’ option added to the default parameters. Only intergenic regions longer than 50 bp were included and Azara serrata Ruiz & Pav.  was included in the alignments as an outgroup. Alignments were used as input on DnaSP 681 to calculate variation between regions. We compared total number of sites, of analyzed positions (NetSites), of segregating sites, of conserved sites, the estimated number of mutations, parsimony informative sites (PIS), proportion of PIS, nucleotide diversity and average number of substitutions per site. The proportion of PIS was calculated as the PIS/NetSites × 100 and gives an estimate about the absolute informativeness of the region. This information, when combined with the length of the region, may be helpful for markers design.To evaluate sequence polymorphism, protein-coding and intergenic regions were extracted with parseGenbank.pl script from MitofyAverrhoa carambola L.  and Elaeodendron orientale Jacq.  were also included as outgroups. A list with all the GenBank accession numbers for additional species is included in the Supplementary Material 50 with the following parameters: -t = d -b5 = a -n = y -e = gb1 -d = y. Therefore, the following analyses were conducted both for the original dataset (nogb) and for the dataset after gblocks (gb). Maximum likelihood inference was conducted based on the concatenated approach for the unpartitioned (CUnogb and CUgb) and partitioned datasets (CPnogb and CPgb) in IQ-tree 282. Models were selected based on Bayesian information criterion (BIC) and support was assessed through 1000 ultrafast bootstraps. For both the nogb and the gb alignments, a total of 25 independent runs were performed for each gene, totaling 2050 consensus gene trees which were included in a single file to generate the 2050 datasets, i.e., 2050nogb and 2050gb. Bayesian Inference for the unpartitioned (CUnogb and CUgb) and partitioned datasets (CPnogb and CPgb) was conducted in Mr.Bayes v.3.2.7a under the best-of-fit model in accordance with BIC assessed in IQ-tree283. It was run for 30 million generations sampled every 1000 generations, using two runs and four chains, until the average standard deviation of split frequencies became less than 0.01, beginning with random trees. The initial trees were discarded after reaching stationarity (~ 25%). Also, a coalescent-based species tree estimation was done in Astral-III84. We conducted the analysis with one tree per gene as input (82gb and 82nogb) and with 25 trees per gene as input (2050gb and 2050nogb). All gene trees used as input in Astral-III84 were rooted and had branches with support lower than 10% collapsed in Newick Utilities85 through the functions nw_reroot and nw_ed. Rooting the trees and collapsing branches with low support are known to improve the performance of summary methods86.For the phylogenetic analyses we assembled a dataset with the 82-protein-coding genes that includes two Bonnetiaceae species (two genera), seven Calophyllaceae species (six genera), four Clusiaceae species (two genera), five Hypericaceae species (three genera) and three Podostemaceae species (three genera). Additionally, data for other 36 species within Malpighiales, 82 for both partitioned and unpartitioned analysis was visualized as a network in SplitsTree4 v. 4.16.287. Additionally, tree conflict was explored through PhyParts51 and pie charts were plotted on the species phylogeny using the PieCharts python script developed by M. Johnson88. PhyParts calculates the number of concordant gene trees, of the top alternative bipartition, of other conflicting topologies, and of uninformative genes for all branches in the tree. To root the trees and remove ultrafast bootstrap and branch length values the files for both PhyParts51 and PieCharts were prepared using Newick Utilities85 through the functions nw_reroot and nw_topology.To explore tree conflict within the clusioid families the splits.nex file generated in IQ-tree2Supplementary Information.


Establishing the different feeding trajectories based on daily enteral feeding data in preterm infants at different gestational ages (GAs), may help to identify the risks and extrauterine growth restriction (EUGR) outcomes associated with the adverse feeding pattern. In a single center, we retrospectively included 625 infants born at 23–30 weeks of gestation who survived to term-equivalent age (TEA) from 2009 to 2020. The infants were designated into three GA groups: 23–26, 27–28, and 29–30 weeks. The daily enteral feeding amounts in the first 56 postnatal days were analyzed to determine the feeding trajectories. The primary outcomes were EUGR in body weight and head circumference calculated, respectively, by the changes between birth and TEA. Clustering analysis identified two feeding trajectories, namely the improving and adverse patterns in each GA group. The adverse feeding pattern that occurred in 49%, 20%, and 17% of GA 23–26, 27–28, and 29–30 weeks, respectively, was differentiated from the improving feeding pattern as early as day 7 in infants at GA 23–26 and 27–28 weeks, in contrast to day 21 in infants at GA 29–30 weeks. The adverse feeding patterns were associated with sepsis, respiratory, and gastrointestinal morbidities at GA 23–26 weeks; sepsis, hemodynamic and gastrointestinal morbidities at GA 27–28 weeks; and preeclampsia, respiratory, and gastrointestinal morbidities at GA 29–30 weeks. Using the improving feeding group as a reference, the adverse feeding group showed significantly higher adjusted odds ratios of EUGR in body weight and head circumference in infants at GA 23–26 and 27–28 weeks. Identifying the early-life adverse feeding trajectories may help recognize the related EUGR outcomes of preterm infants in a GA-related manner. The advances in medical care in the neonatal intensive care unit (NICU) has resulted in an increasing survival rate of extremely preterm infants. Studies have demonstrated the importance of adequate feeding, nutrition and growth, especially in this preterm population ,2. EarlyGrowth velocities of 14–20 g/kg/day using volumes of approximately 120–150 mL/kg/day of fortified human milk or preterm formula have been recommended to provide adequate growth rates ,6,7. HowMany studies have focused on the relationship between early feeding, protein and caloric intake and postnatal growth outcome in preterm infants ,3,8,9,12Longitudinal data are data in which each variable is measured repeatedly over time. One method for analyzing longitudinal data is clustering analysis. The kmlShape, a method for data partitioning, provides clusters on the basis of trajectory shape; kmlShape analysis allows for the grouping of individuals whose trajectories have similar forms, albeit with a shifting of positions over time . This anThis study enrolled 717 very preterm infants who were born between 23 and 30 weeks of gestation and admitted within three days after birth to the tertiary NICU of this university hospital from January 2009 to October 2020. This study was approved by the Institutional Review Board of National Cheng Kung University Hospital .The 625 infants (87%) who survived to term-equivalent age (TEA)  were included for analysis. The infants were designated into three populations: GA 23–26 weeks, 27–28 weeks, and 29–30 weeks. This study was carried out in a 20-bed tertiary neonatal intensive care unit (NICU) at the National Cheng Kung University Hospital in Tainan, Taiwan. Approximately 350 neonates were admitted to the unit per year, including 60–80 very preterm infants. A body weight measurement was usually performed daily during morning care as a baseline for prescribing pharmacy dosages and milk volumes. Based on the feeding protocol of preterm infants in this university hospital, very preterm infants are initiated with parenteral and enteral nutrition soon after birth . If the The information of daily enteral feeding data and body weight in the first 56 postnatal days were retrieved from an electronic medical system and presented as mL/kg/day, calculated based on the body weight measured on the day. Within each GA group, the feeding trajectories were analyzed based on the daily enteral feeding amounts using the “kmlShape” package in R to cluster meaningful groups .Demographics, perinatal and neonatal risk factors, neonatal morbidities and GI morbidities requiring surgery were reviewed by a case manager and two neonatologists . NeonataWe recoded the anthropometric measurements for body weight and head circumference at birth and at TEA . EUGR wap < 0.05 was considered statistically significant. Demographic data and perinatal and neonatal risk factors were compared among the three GA preterm groups using chi-square or Fisher’s exact tests for categorical variables, and analysis of variance or Kruskal–Wallis tests for continuous variables. The logistic regression model was applied to identify the risk factors and morbidities that might affect these feeding trajectories. Using logistic regression and adjusting for the selected risk factors, the association between the feeding trajectory pattern and EUGR outcomes was analyzed. All candidate covariates were selected by the p-value of less than 0.1 from univariate analysis. After univariate analysis, all candidate factors were included in the multivariable analysis and were chosen by the stepwise procedure with the Akaike information criterion. A value of Of the 625 infants (87%) included for analysis, 183 infants (29%) were at GA 23–26 weeks, 215 infants (35%) at GA 27–28 weeks, and 227 infants (36%) at GA 29–30 weeks. Among the three GA preterm groups, there were significant differences in the demographic risks, respiratory/hemodynamic morbidities, and GI morbidities. The smaller the GA group, the older the postnatal age when trophic feeding was started, and full feeding was reached . The kmlp < 0.01), and the differences increased from days 7 to 56  (p < 0.001), and the differences increased throughout days 7 to 56  (p < 0.05) when a late deterioration occurred, and the differences remained significant at days 28, 35, 42, and 56  and adverse feeding in 89 infants (49%). Compared to the improving feeding group, the adverse feeding group was on a significantly smaller daily median milk volume by postnatal day 7 (< 0.001) A. In inf< 0.001) B. In inf, and 56 C. Compared to the improving feeding group, the adverse feeding group was significantly lower in gestational age, had higher rates of respiratory/hemodynamic morbidities, including hypotension requiring vasopressors, hs-PDA requiring surgery, late-onset sepsis and a longer duration requiring IMV, and GI morbidities, such as non-NEC events requiring surgery in infants at GA 23–26 and 27–28 weeks. The adverse feeding group also had significantly higher rates of NEC GI morbidities, including stage 1 and severe NEC in infants at GA 27–28 and 29–30 weeks .Using the improving feeding trajectory as a reference, univariate logistic regression analysis  followedAt TEA, compared to the improving feeding group, the adverse feeding group had significantly higher rates of EUGR in body weight (∆z < −1) in the three preterm groups, and severe EUGR in body weight (∆z < −2) in preterm infants at GA 23–26 and 27–28 weeks . The advThe close monitoring of the respective feeding patterns of preterm neonates in each GA preterm population was essential to identify any deviations from normal growth patterns. In this study, we established improving versus adverse feeding patterns by clustering analysis in each of the three different GA preterm populations. Relative to the improving feeding pattern, the adverse feeding pattern occurred in almost 50% of infants at GA 23–26 weeks, and 17–20% of infants at GA 27–28 and 29–30 weeks. The adverse feeding pattern could be distinguished from the improving pattern as early as postnatal day 7 in the two extremely preterm GA groups, but as late as day 21 in the very preterm group, which was significantly related to the shared and distinct respiratory/hemodynamic and GI morbidities that occurred in each preterm group. In addition, the smaller the GA group, the larger the impact of the adverse feeding pattern on EUGR outcomes in body weight and head circumference. These findings suggested that establishing feeding trajectories in different GA preterm groups could early identify infants at risk of worse growth outcome in body weight and head circumference at TEA, particularly in extremely preterm infants. Feeding difficulties in preterm infants, and the consequent effects on EUGR rates, are already known and well described in the literature. This study is not only the first to establish different feeding trajectories based on data for daily enteral feeding amounts in preterm infants at different GA groups, but it also identified the specific risks and morbidities that are related to adverse GA feeding patterns.Preterm infants with EUGR are considered to be at risk of abnormal growth and neurodevelopment outcomes in childhood ,30. The We categorized preterm infants into three different GA groups and revealed that the two different feeding patterns in each GA group had a differential association with EUGR in terms of body weight and head circumference, defined by the Δz-scores between birth and TEA. The EUGR in both body weight and head circumference occurred simultaneously only in infants at GA 23–26 and 27–28 weeks, but not in infants at GA 29–30 weeks. In-hospital head circumference growth could be more accurate than body weight to predict neurodevelopmental outcomes . TherefoMonitoring the changes in daily median feeding amount could provide valuable information for early identification of extremely preterm infants who are likely to develop adverse feeding trajectories as early as postnatal day 7. In contrast, the feeding patterns of very preterm infants at GA 29–30 weeks were quite different to those of extremely preterm infants. In this GA group, infants had similar feeding patterns until day 21, when feeding deterioration occurred due to the onset of NEC in this preterm population. These findings suggested that identifying infants who will follow an adverse feeding trajectory early by the daily feeding amount is more applicable to extremely preterm infants. The feeding pattern of progression followed by late deterioration was distinctive for the very preterm infants. Respiratory/hemodynamic morbidities, such as RDS, hs-PDA, and IMV use, have been reported to be related to feeding intolerance in preterm infants ,34,35,36The introduction and advancement of enteral feeds for preterm infants are often delayed because of concerns that early full enteral feeding will not be well tolerated or may increase the risk of NEC ,6. Our fFeeding trajectories are important not only for nutritional support but also for the growth outcomes of preterm infants. In this study, we defined EUGR by the z-score changes in body weight and head circumference between birth and TEA instead of at discharge or PMA 36 weeks, as frequently used by others ,33. We dData for daily enteral feeding amounts are valuable for preterm infants. Establishing the GA-related feeding trajectories based on these data for preterm infants may lead to early identification of morbidities. Close monitoring of different feeding patterns is important for early mitigation with GA-related morbidities, in order to reverse adverse feeding trajectories, thereby reducing EUGR outcomes for vulnerable infants.


Animals of the control group (n = 7) did not receive any resuscitation regime. The interventional groups were treated exclusively with either crystalloid (n = 8) or continuous norepinephrine infusion (n = 7). Results: Haemorrhagic shock led to a drop in tissue oxygenation parameters in all groups. These correlated with established indirect markers of tissue oxygenation. Fluid therapy restored tissue oxygenation parameters. Skin and kidney measurements correlated well. High dose norepinephrine therapy deteriorated tissue oxygenation. Tissue water content increased both in the skin and the kidney in response to fluid therapy. Conclusions: HSI detected dynamic changes in tissue oxygenation and perfusion quality during shock and was able to indicate resuscitation effectivity. The observed correlation between HSI skin and kidney measurements may offer an estimation of organ oxygenation impairment from skin monitoring. HSI microcirculatory monitoring could open up new opportunities for the guidance of haemodynamic management.Background: The ultimate goal of haemodynamic therapy is to improve microcirculatory tissue and organ perfusion. Hyperspectral imaging (HSI) has the potential to enable noninvasive microcirculatory monitoring at bedside. Methods: HSI (Tivita Haemodynamic therapy aims to maintain tissue perfusion and oxygen delivery to protect organ function during phases of circulatory compromise . A prere® Tissue camera system  provides noninvasive HSI for qualitative and quantitative bedside microcirculatory assessment. The key feature of this HSI camera system is a set of four parameters that allow a differentiated and spatially resolved investigation of tissue oxygenation, perfusion quality, haemoglobin distribution, and tissue water content [Hyperspectral imaging (HSI) is an evolving noninvasive imaging technology that provides an evaluation of intrinsic biochemical tissue characteristics based on tissue-light interactions . HSI tec content ,8. Studi content ,10. Firs content .In a double-hit porcine model of major abdominal surgery and haemorrhagic shock, we aimed to evaluate whether HSI can detect pathological skin and kidney tissue oxygenation and perfusion deficits, the response to fluid and vasopressor therapy, and detrimental resuscitation side effects. Furthermore, correlations between skin and kidney HSI findings, as well as correlations to macrohaemodynamic variables and global markers of tissue oxygenation, should be evaluated.This study implemented on 27 pigs (Sus scrofa) was approved by the appropriate governmental body  and conducted in accordance with the European law on the protection of animals used for scientific purposes (EU-Directive 2010/63). The study reporting adheres to the ARRIVE guidelines 2.0 .Serum lactate after one hour of therapy following shock induction was used as the effect variable for sample size calculation. A decrease of 25%, compared with the control group, was considered biologically and clinically relevant. For calculations , a standard deviation of 20%, a significance level of α = 0.05, and a power of 1-β = 0.8 were applied, resulting in an estimated sample size of 8 animals per group. There was no preferred sex. All animals were assigned to the treatment groups in advance. The investigator who selected the animals for the experiment was not aware of the group assignment. Experimenters were not blinded to the group assignment.® 5 mg/mL by hameln pharma plus Gmbh®, Hameln, Germany) and 10 mg/kg ketamine . Pre-medication was administered by intramuscular injection to the neck. In unconscious pigs, an auricular vein catheter was placed. For endotracheal intubation , the animal was placed in a prone position, and 2 mg/kg of propofol was administered. Throughout the experiment, anaesthesia was maintained with a combination of intravenous 0.5–1 mg/kg/h midazolam, 10–20 mg/kg/h ketamine, and inhalational Sevoflurane® (exsp.vol% 1.5–2.5). Depth of anaesthesia was confirmed regularly by the lack of spontaneous movements and absence of reaction and cardiovascular signs to surgery or painful stimulation between the front hooves.All pigs were kept inside in the Interfacultary Biomedical Facility of the University of Heidelberg at a constant temperature and a controlled circadian rhythm. They fasted the day of surgery. Water was accessible until anaesthesia induction. The pigs were premedicated with 1 mg/kg midazolam  with a tidal volume of 8 mL/kg, a positive-end-expiratory pressure of 5 mbar, an inspiration-to-expiration ratio (I:E ratio) of 1:2, and a fixed FiO2 of 0.5 was performed. The respiratory rate was adjusted to reach an end-tidal CO2 of 40 ± 5 mmHg. Peripheral oxygen saturation was monitored with a saturation probe fixed to the tail.Volume-controlled ventilation  and an arterial PiCCO catheter  were implemented to allow continuous haemodynamic monitoring including heart rate, blood pressure, stroke volume variability, cardiac output measurement  with a temperature of 4 °C, and blood gas analysis. Body temperature was sustained with electrical heat blankets and monitored with an oesophageal temperature probe.The right external jugular vein and the right femoral artery were surgically prepared. A central venous catheter  of 40 ± 5 mmHg for 60 min.® by B. Braun®, Melsungen, Germany). The control group (CG) did not receive a specific haemodynamic treatment regime following shock induction. The fluid-treated group (FG) received a continuous crystalloid infusion . The norepinephrine-treated group (NG) received a continuous infusion of noradrenaline . In both FG and NG, a MAP of 65 mmHg was targeted for the first treatment period of 60 min and a MAP of 90 mmHg for the second treatment period of 60 min . The technical specifications of the system have been described in previous publications [-2, wavelength range: 500–650 and 700–815 nm): haemoglobin oxygen saturation in the capillary system of superficial tissue (penetration depth up to 1 mm) indicated in percent (0–100%);Tissue oxygenation : haemoglobin oxygen saturation in the capillary system of deeper tissue layers (penetration depth up to 4–6 mm) indicated in predefined arbitrary units (0–100);-Tissue haemoglobin index : distribution of deoxygenated and oxygenated haemoglobin in the measured tissue (penetration depth up to 1–3 mm) indicated in predefined arbitrary units (0–100);-Tissue water index : relative water content of the tissue (penetration depth up to 1–3 mm) indicated in predefined arbitrary units (0–100).Hyperspectral imaging (HSI) was performed with the Tivitaications ,8,9. The®TM Suite) was used to define a circular region of interest (ROI) in each HSI image to obtain objective numerical values. One unit of the circular ROI equals the radius in pixels. The largest possible circular area of homogenous tissue uninterrupted by any lesions, plicae, or light reflections was selected. ROI size at the ear and the kidney was 30 and 20 arbitrary units (HSI measurements were conducted on the inside of one ear (skin measurement site) and the right kidney. The camera-specific software . GraphPad Prism  was used for statistical analyses. Descriptive statistics were performed for the complete dataset. Mean and standard deviation are presented. Spearman’s correlation analyses were used. Metric data over courses of time and between intervention groups were evaluated by analyses of covariance with a mixed effect model. For the comparison of paired samples, the Wilcoxon test was used.Data were collected with the aid of an electronic database system  were assessed in the study, and data from 22 animals  were analysed. Three pigs died during the observation period, and two were excluded from the analysis due to severely impaired haemodynamics at baseline.2 gap, oxygen extraction, and lactate increased, while central venous oxygen saturation (ScvO2) decreased , the MAP targets of 65 mmHg in the first and 90 mmHg in the second hour of intervention were not achieved A.2 .TWI of the skin and the kidney increased G,H and c2 gap and lactate increased dose-dependently in the NG, whereas no differences in ScvO2 and oxygen extraction between NG and CG were observed  induced a persisting rise in heart rate, whereas MAP fell again after a transient increase at 30’ after intervention start A. Noradrobserved E–H.2 of the skin and the kidney, as well as skin NIR  and distribution of oxy-and deoxygenated haemoglobin (THI). Our group observed that septic nonsurvivors had significantly elevated THI on admission to the intensive care unit—these patients showed increased vasopressor dependency [The administration of norepinephrine without an additional fluid resuscitation in the NG did not result in attaining MAP targets despite steady dose escalations. Moreover, HSI tissue oxygenation parameters StOerfusion . We obseerfusion ,20. HSI pendency . Previoupendency . We proppendency .2 and NIR showed a parallel decrease at the skin and kidney in the NG. One possible interpretation could be that the increased tissue haemoglobin on the skin reflects reduced capillary efflux with consecutive capillary congestion. In combination with low tissue oxygenation parameters, THI could be useful in assessing perfusion quality and potentially warn physicians of the detrimental effects of excessive catecholamine administration. The correlation of oxygenation parameters between the skin and the kidney measurement site was stronger under norepinephrine than fluid treatment. The reason for that could lie in compensatory mechanisms of centralisation, in which central organs are preferentially supplied with blood during shock. Administration of norepinephrine could affect these mechanisms by altering the perfusion distribution between peripheral and organ tissues, thereby worsening perfusion quality in both tissues equally, resulting in a better correlation and high haemoglobin content of the skin [In this study, we observed that skin and kidney THI showed no relevant changes during haemorrhagic shock induction. In the NG, THI revealed opposite behaviour following norepinephrine administration: skin THI increased and kidney THI decreased, whereas StOthe skin .Fluid overload and concomitant tissue oedema formation are dreaded detrimental effects of fluid therapy ,23. Ther2 and NIR. THI and TWI could potentially be useful to warn of detrimental effects of vasopressor and fluid therapy, respectively. The combined evaluation of tissue oxygenation, haemoglobin, and water content could open up new opportunities for the guidance of haemodynamic management, which has to be substantiated in future interventional trials.HSI detected dynamic changes in tissue oxygenation and perfusion quality during shock and was able to indicate resuscitation effectivity. Further, HSI measurements of skin oxygenation reflected the situation in the kidney well and thus support the hypothesis to monitor skin microcirculatory alterations during circulatory compromise to infer organ oxygenation adequacy. We propose that distinctive HSI parameter patterns could enable tissue-perfusion-guided therapy targeting StO


The use of antimicrobials in animal production is under public debate, mainly due to the risk of transfer of resistance to pathogenic bacteria in humans. Therefore, measures have been taken during the last few decades to reduce antibiotic usage in animals, for instance, by national monitoring programmes and by improving animal health management. Although some initiatives exist in molecular genetic selection, quantitative genetic selection of animals towards decreased antibiotic usage is an underexplored area to reduce antibiotic usage. However, this strategy could yield cumulative effects. In this study, we derived new phenotypes from on-farm parenteral antibiotic records from commercially grown crossbred finishing pigs used in the progeny test of Piétrain terminal sires to investigate the heritability of antibiotics usage. Parenteral antibiotic records, production parameters and pedigree records of 2238 full-sib pens from two experimental farms in Belgium between 2014 and 2020 were analysed. Heritability estimates were moderate (18–44%) for phenotypes derived from all antibiotic treatments, and low (1–15%) for phenotypes derived from treatments against respiratory diseases only. Moreover, genetic correlations between these new phenotypes and mortality were low to moderate (0.08–0.60) and no strong adverse genetic correlations with production traits were found. The high heritabilities and favourable genetic correlations suggest these new phenotypes, derived from on-farm antibiotics records, to be promising for inclusion in future pig breeding programs to breed for a decrease in antibiotics usage. Antimicrobials are commonly used in animal production systems, not only for treatment, but also for metaphylaxis, prophylaxis and in some countries still for improvement of feed efficiency and growth ,2. HowevReducing antibiotic usage in animals by genetic selection has been underexplored so far . BreedinIn this study, we investigate the value of field data on antibiotic usage in finishing pigs on a pen level as a new, easy to measure phenotype in pig breeding. Genetic parameters such as heritability and genetic correlations were estimated for antimicrobial usage in finishing pigs.Mycoplasma hyopneumoniae before weaning and received anthelminthic medication upon arrival at the experimental farm. Pigs were kept in commercial conditions representative for Belgian pig production  but were kept in pens of only 6 to 7 full-sib pigs. Farm A had 18 compartments, each containing 8 pens (4.7 m2 per pen) of usually 6 pigs. Pens in farm A were of mixed sex with females and immunocastrated males to prevent boar taint . Farm B had 7 compartments, each with 16 pens (5.4 m2) of usually 7 pigs. Pens in farm B were of mixed sex composed of females and surgically castrated males (barrows). Only pens starting with 5 to 8 full-sib pigs were retained for further analyses.Data were collected on crossbred progeny of Piétrain sires and hybrid sows  kept in two experimental fattening units (farm A and B) of the breeding organization “Vlaamse Piétrain Fokkerij” . After quality control, data were available on 2238 full-sib pens with known pedigree  and treatment duration (days). Other medication such as vaccines, anthelmintic products and anti-inflammatory drugs were not considered in this study.Aside from antimicrobial usage, feed intake (FI) during the testing period was also recorded at the pen level. Average daily gain (ADG), mortality during the finishing period and carcass quality traits (meat%) were collected at the individual level. Pigs that died before slaughter were individually weighed so that all weight data could be adjusted and retained in the analysis.As the antimicrobial usage data in our study were collected at pen level, these data were summarised and analysed per pen. In total, 779 pens were medicated at least once. In farm A, however, 104 pens  were group medicated at the compartment level with an orally applied antibiotic . These pens were excluded due to the different route of administration  of the antibiotic.all) and another set with antibiotic treatments for respiratory problems only (ABrespiratory). The ABrespiratory dataset was created because the majority of antibiotics were used to treat respiratory diseases . In ABrespiratory, treatments with fluoroquinolones (1 pen) and macrolides (7 pens) were rare and therefore these treatments were removed from the dataset. An overview of the antibiotics reported in this study is shown in Hereafter, two datasets were created: one dataset containing all antibiotic treatments (ABtreatment) in a pen was calculated as the treatment duration in days for a specific treatment.The treatment length (NmL) was calculated for each treatment by multiplying the administered volume per pig by the number of treated pigs in the pen and the treatment duration:The total administered volume of an antibiotic (ABmg) was computed afterwards, using the concentration (mg/mL) of the active compound in the administered product , as suggested by Timmerman et al. .Pen; mg/kg) was calculated as the mg of active compound administered per kg of live weight of pig in the pen at the time of administration UDPen (t is the time at treatment (age in days). The growth curve parameters were estimated using the available weight data at start and at death or slaughter (The weight at treatment was estimated based on the age of pigs using a Gompertz growth curve function:laughter .pig) and animal daily dose per pig (ADDpig) were calculated following Timmerman et al. [pig is the administered dose of a drug per kg pig per day, whereas the ADDpig is the nationally recommended dose of a drug per kg pig per day. These parameters were used to assess whether dosing had been done according to label directions. Antibiotic administrations where pig were based on dose recommendations by the Belgian expertise center on antimicrobial use in animals .ADD and TIUDD) were calculated at the pen level for each parenterally administered treatment [mg; UDD or ADD is as specified before; LA is a correction factor for long acting antibiotics [ADD and TIUDD can be interpreted as the percentage of days in a period ‘at risk’ a pig is treated with one reference dose (ADDpig or UDDpig). For example, a TIUDD of 1 indicates that an average pig was treated with one reference dose of antibiotics for 1% of the period at risk.Finally, treatment incidences , whereas traits in the ABrespiratory dataset received a suffix ‘-respiratory’ .The parameters NAverage daily gain (ADG) was calculated individually as the difference between weight at slaughter/death and start of test (kg), divided by duration of test (days). The percentage of lean meat (meat%) was estimated using a fully automated classification system  at two slaughterhouses from the Belgian Pork Group. Mortality counts were summed at the pen level. Feed conversion ratio (FCR) was calculated as the mean FI (kg/day) divided by the mean ADG (kg/day) per pen. Details of trait distributions are shown in The final dataset after data cleaning included 2238 pens: 14,742 finishing pigs from 699 Piétrain sires and 1148 crossbred dams. Pedigree comprised 24,081 animals, where the median pedigree depth of Piétrain sires was 11 generations  and 5  for crossbred dams.2) were estimated using single-trait genetic animal models with average information REML, implemented in airemlf90 and invoked with the R-package breedR [g) between traits related to antibiotic usage  were estimated using bivariate animal models (airemlf90). The h2 was estimated as the proportion of additive genetic variance divided by total variance, whereas c2 was estimated as the proportion of variance explained by random environmental effects (c), divided by total variance. Standard errors of h2, c2 and rg were estimated using the se_covar_function option in airemlf90.Heritabilities  was fitted on the complete datasets. From these results, observed phenotypes were adjusted for fixed and non-genetic random effects: nt-level .all dataset). Most of the 1538 antibiotic treatments were from the class of lincosamides (44.5%), cephalosporins (18.4%), aminopenicillins (18.3%) and florfenicol (11.4%). The majority of treatments were applied for respiratory diseases (70.5%). Other indications included locomotion problems (8.7%), diarrhoea (5.6%), severe weight loss (4.5%), oedema disease (3.6%), ear infections (2.9%) and others (4.2%). The number of treatments per month is given in In total, 626 pens had a minimum of one pig treated, at least once, with antibiotics  and mortality were zero-bound and showed a heavy right-tailed distribution. Other traits were approximately normally distributed based on visual inspection of trait distributions.Descriptive statistics of the studied traits at a pen level are shown in all dataset, pens were treated 0.7 times with 3.6 mL of antibiotics and 445.1 mg of active compound , whereas 49.4% of treatments were underdosed and 17.1% were overdosed. Treatment incidences  ranged between 0.08 and 0.17, indicating that an average pig was treated with one reference dose of antibiotics for 0.08–0.17% of the period at risk (119 days median).On average for the ABcompound . The mea2, c2 and rg are shown in mg, TIADD and UDpen were most heritable and significantly >0 based on a 95% confidence interval (mean ± 1.96×se), which is not explicitly given. In general, c2 was moderate to high for antibiotic usage (12–38%) and estimates were larger in the respiratory problems dataset. UDpen and Ntreatment across datasets were highly genetically correlated (rg = 0.65 to 0.95). Genetic correlations with production traits were low with high standard errors, but the genetic correlation with mortality was consistently positive, ranging from 0.08 to 0.60.Estimates of hpen and Ntreatment are shown in respiratory dataset using the across-family sampling strategy. Within-family sampling yielded—as expected due to the exploitation of close relationships—higher predictive abilities with lower standard errors. Predictive ability of UDPen was higher than for Ntreatment. Heritabilities obtained in cross-validations were similar to the full dataset.Results of cross-validation analyses for UDThis study is the first to report genetic parameters of antibiotic usage derived from medication records in finishing pigs. New phenotypes for antibiotic usage at a pen level in finishing pigs were derived from on-farm medication records and were found to be moderately heritable and with a favourable (low to moderate) genetic correlation with mortality.mL, ABmg, Ntreatment, TIADD, TIUDD and UDPen. Data were summed over different antibiotics at a pen level with an equal weight to the administered mg of active compound, although potency (strength) of active compound can differ among products [Six different phenotypes derived from medication records at pen level were investigated: ABproducts . Howeverpig/ADDpig ratios showed that most treatments were underdosed (61.9%), while 12.6% were overdosed and 25.5% correctly dosed  were low, ranging from 0.09 to 0.15 units, meaning that pigs were treated with one reference dose of antibiotics for 0.09–0.15% during the period at risk (fattening period). Sjölund et al. [ADD and TIUDD are thus rather low, especially compared to the median Belgian value of 0.83.Treatment incidences  and/or ordinal traits , assuming that a higher health status would decrease antimicrobial usage and mortality ,30,31. F2) estimates for traits related to antibiotic usage were moderate to high in the dataset containing all parenteral antibiotic treatments (18–44%) and low to moderate in the respiratory problems dataset (1–15%)  or mL product (ABmL) might be interesting as description for the used antibiotics, however it seems less relevant for estimates of h2: 10 mg (or mL) of antibiotic x is not necessarily equivalent to 10 mg (or mL) of antibiotic y. Therefore, UD and/or TIADD seem better parameters for quantifying antibiotic usage, since they take into account the mg of active compound administered per kg of animal at risk.Heritability (h (1–15%) . The h2 2 estimates were robust against random sampling of data and also the reduction in data points.Heritability estimates remained relatively consistent in the cross-validations , implyintreatment-all and Ntreatment-respiratory were 25% and 3%, respectively. Putz et al. [Heritability estimates of Nz et al.  found a z et al.  found hez et al. ).2) were moderate to high for traits related to antibiotic usage. This was expected since disease prevalence and antimicrobial usage are known to show seasonal variation in pigs, depend on the presence of infectious agents and are linked to the stable climate [2-estimates were consistently higher in the respiratory problems dataset, meaning these environmental effects within a compartment are larger for respiratory problems. Indeed, the stable climate is well known to play a pivotal role for the respiratory health of pigs [Common environmental effects  were exploited. This indicates the potential of UDPen for use in pig breeding programs. Applying one generation of single-trait selection to reduce UDPen-all (additive genetic variance is 3.1 mg2 active compound/kg2 pig) with a selection intensity of 1, a generation interval of 3 years and an accuracy of 0.50 would yield an annual reduction of UDPen-all by 0.29 mg active compound/kg pig when using the breeder’s equation and assuming an equal potency between products.Predictive abilities using five-fold cross-validation were highest and significantly larger than zero for UDPen and Ntreatment with mortality at pen level were low to moderately high , mortality at the pen level would simultaneously decrease as a correlated response. We believe this is an extremely interesting result, as mortality is one of the most important and ignored traits in pig breeding programs. Furthermore, no strong adverse genetic correlations were found with production traits such as FI, ADG and FCR. However, we would like to note that not all genetic correlations were significantly different from zero. Only between Ntreatment and Meat% unfavourable genetic correlations were found (0.45 and 0.11), whereas low favourable genetic correlations were found between UDpen and ADG (−0.30 and −0.21) and FCR (0.25 and 0.26). These results indicate that UDPen or Ntreatment can be included in current breeding programs without adversely affecting other economically important traits. Moreover, breeding pigs for lower UDPen or Ntreatment will not only decrease antimicrobial usage by increasing general pig health, but may also decrease costs  for pig farmers and reduce the risk of antibiotic resistance [Genetic correlations of UD.60; Pen . This gesistance .The present study is based on data from finishing pigs at pen level in two experimental farms considered representative for the Belgian pig sector. The dataset contained 2238 full-sib pens, which is a reasonable size to estimate heritabilities, but might still be insufficient to accurately estimate genetic correlations although standard errors of genetic correlations were low to moderate . FurtherIn the present study, data on antibiotic usage were recorded on paper. However, recent technology allows automated recording of treatments of individual pigs in real time using RFID tags. Such new precision livestock farming solutions may offer a user-friendly solution to record antibiotic usage in large scale pig breeding programs. Individual data may increase accuracy of breeding values and, hence, genetic progress. Moreover, individual recording is required in pens of non-siblings in order to exploit pedigree or genomic relationships.Individual tracking using RFIDs would also allow to study the suckling and weaning periods during which most antibiotics are used. Sarrazin et al.  and SjölThis study shows that new phenotypes, derived from on-farm drug records at pen level from Piétrain-sired crossbred finishing pigs in a commercial environment, were heritable. Moreover, low to moderate favourable genetic correlations were found with mortality and  favourable genetic correlations were estimated with production traits such as average daily gain and feed conversion ratio. Hence, these new phenotypes are promising traits to be included in the pig breeding programs, although more research is still necessary at this point. Further improvements are expected by recording antibiotic usage on an individual level throughout a pig’s lifespan via emerging precision livestock farming technologies.