Enterocytozoon bieneusi, an obligate intracellular microsporidian parasite, can infect humans and a wide variety of animals worldwide. However, information on the prevalence and molecular characterization of E. bieneusi in pet rats and guinea pigs is lacking. In this study, 325 fecal samples were collected from 152 pet fancy rats and 173 pet guinea pigs purchased from pet shops in Henan and Shandong provinces. The prevalence of E. bieneusi was 11.2% (17/152) in pet fancy rats and 20.2% (35/173) in pet guinea pigs. Genotypes D (n = 12), Peru11 (n = 3), S7 (n = 1) and SCC-2 (n = 1) were identified in pet fancy rats, and genotype S7 (n = 30) and a novel genotype PGP (n = 5) were identified in pet guinea pigs. The ITS sequence and its phylogenetic analysis showed that the novel genotype PGP was distinctly different; it exhibited less than 50% similarity to the reference sequences, and did not cluster with any of the known E. bieneusi genotype groups, forming a unique branch between groups 6 and 7. These data suggest that this is a new E. bieneusi genotype group. This is the first report of E. bieneusi infection in pet fancy rats and pet guinea pigs worldwide. The identification of zoonotic genotypes D, Peru11, and S7 suggests that pet fancy rats and guinea pigs can be potential sources of human microsporidiosis. Enterocytozoon bieneusi, a unicellular and obligate intracellular pathogen, has an extensive host range and has been identified in humans, livestock, companion animals, and wildlife, as well as in wastewater [Enterocytozoon bieneusi infection can cause self-limiting diarrhea, malabsorption, and wasting in immunocompetent hosts and life-threatening diarrhea in immunocompromized individuals [stewater , 23. Entividuals . Humans ividuals .E. bieneusi from various hosts [E. bieneusi genotypes have been identified in rodents worldwide [Rattus spp.), only three surveys have focused on the molecular characterization of E. bieneusi in wild rats in Iran and China, and six genotypes  have been identified [Genotyping based on the internal transcribed spacer (ITS) region of the rRNA gene has identified 11 major phylogenetic groups and more than 470 genotypes of us hosts . To dateorldwide , 30, 34.entified , 30, 34.Rattus norvegicus forma domestica, are rodents belonging to the order Rodentia and family Muridae. Fancy rats have been bred as pets at least since the late 19th century; they are considered to be intelligent, playful, and trainable animals (http://www.afrma.org/). In recent years, fancy rats have become a very popular pet in China. Pet rodents can be hosts to several zoonotic pathogens, including viruses, bacteria, and parasites [E. bieneusi to a child from household guinea pigs has been reported [E. bieneusi in pet rats and pet guinea pigs. Therefore, the aim of the present study was to determine the prevalence and genotypes of E. bieneusi in these animals and to assess its zoonotic potential.Fancy rats, arasites ; zoonotireported . HoweverThe research protocol was reviewed and approved by the Research Ethics Committee of Henan University of Science and Technology.Between September 2018 and October 2019, 152 pet fancy rats and 173 pet guinea pigs were purchased from six pet shops in Luoyang, Henan and Weifang, Shandong, China  and 2. Ug at room temperature. Before DNA extraction, 200 mg of each fecal sample was added to a 2 mL microcentrifuge tube containing 200 mg of glass beads, and were vortexed at maximum speed until the fecal samples were completely homogenized. Genomic DNA was extracted using an E.Z.N.A. Stool DNA Kit , according to the manufacturer’s instructions. The extracted DNA was kept at −20 °C before being used in PCR analysis.Each specimen was washed with distilled water by centrifugation for 10 min at 3000 ×Enterocytozoon bieneusi was examined by nested PCR targeting a ~390-bp fragment of the ITS region, as previously described [® Taq DNA Polymerase  was used for PCR amplifications. The cycling conditions for PCRs were: 94 °C for 5 min; followed by 35 cycles of 94 °C for 30 s, 57 °C (primary PCR) or 55 °C (secondary PCR) for 30 s, and 72 °C for 40 s; followed by 72 °C for 7 min. Positive and negative controls were included in each PCR analysis.escribed . The prihttp://www.clustal.org/) [E. bieneusi were determined based on ~243 bp of the ITS region, according to the established nomenclature system [http://www.megasoftware.net/) [Two-directional sequencing of positive PCR products was done by Sangon Biotech Co. Ltd., . The obtained nucleotide sequences were aligned with available sequences in GenBank, using ClustalX 2.1  . Genotype system . A neighre.net/) . The evoE. bieneusi and the age, sex, and region of pet fancy rats and guinea pigs using SPSS, version 17.0 .Chi-square analysis was performed to assess the correlation between the prevalence of E. bieneusi obtained from pet fancy rats and guinea pigs in this study were deposited in the GenBank database under accession numbers MN550998–MN551001 and MN998614–MN998615, respectively.Unique ITS nucleotide sequences of E. bieneusi was detected by PCR in 17 (11.2%) of 152 pet fancy rats and 35 (20.2%) of 173 pet guinea pigs. To our knowledge, this is the first report of E. bieneusi infection in pet rats and pet guinea pigs worldwide. To date, there have been three studies focusing on E. bieneusi infection in wild rats in Iran and China [E. bieneusi in pet fancy rats was slightly higher than that (4.0%–8.9%) in wild rats in the above-mentioned reports. The prevalence of E. bieneusi in pet guinea pigs in this study was higher than that  in household guinea pigs in Peru [In the present study, nd China , 30, 34 hat 4.0%–.9% in wi in Peru .E. bieneusi in younger animals and those from Weifang, Shandong were higher than those in older animals and animals from Luoyang, Henan (p > 0.05). This finding was consistent with the observations reported in a previous study on pet red-bellied tree squirrels in China [In both pet fancy rats and guinea pigs, although the prevalences of g, Henan  and 2, tin China .E. bieneusi ITS-positive samples from pet fancy rats in this study, four known genotypes were identified; genotype D (n = 12) was the dominant genotype, followed by Peru11 (n = 3), S7 (n = 1), and SCC-2 (n = 1)  and a novel genotype  (E. bieneusi in rats have been limited to three reports in wild rats (R. norvegicus and R. rattus) in Iran and China, and a total of six genotypes were identified, including genotypes D, CD6 (synonyms: CHG14), Peru6, M, BEB6, and CHG2 [In the 17  (n = 1) . For pet, n = 5) . Until nand CHG2 , 30, 34 Callosciurus erythraeus) (75.0%), pet red squirrels (Sciurus vulgaris) (44.3%), and domestic bamboo rats (Rhizomys sinensis) (77.3%) [In this study, genotype D was the most prevalent genotype in pet fancy rats, which is consistent with two previous reports (85.7% and 89.5%) from wild rats , 34, as  (77.3%) , 7, 26.  (77.3%) . In Chin (77.3%) –29, 32.  (77.3%) , 33, 34.In the present study, genotype Peru11, a zoonotic genotype, was identified in pet fancy rats for the first time. This genotype has been found previously in humans in Peru, China and Thailand, non-human primates in Kenya and China, raccoons, voles and cottontails in the United States, chickens in Brazil, cats in Spain, and minks and water in China , 17, 33.E. bieneusi ITS region  were identified in this study, and genotypes D and S7 were the dominant genotypes in pet fancy rats and guinea pigs, respectively. Rats (Rattus norvegicus) are a new host of E. bieneusi genotypes Peru11, S7, and SCC-2, and guinea pigs might be an important reservoir host of genotype S7. The identification of three zoonotic genotypes  suggests that pet fancy rats and guinea pigs may be the sources of E. bieneusi infection in humans. Therefore, pet owners, especially children, should be educated to take precautions to reduce the transmission risk.This is the first report of The authors declare that they have no competing interests.


The test–retest reliability of the one-repetition maximum (1RM) test varies across different studies. Given the inconsistent findings, it is unclear what the true reliability of the 1RM test is, and to what extent it is affected by measurement-related factors, such as exercise selection for the test, the number of familiarization trials and resistance training experience.The aim of this paper was to review studies that investigated the reliability of the 1RM test of muscular strength and summarize their findings.The PRISMA guidelines were followed for this systematic review. Searches for studies were conducted through eight databases. Studies that investigated test–retest reliability of the 1RM test and presented intra-class correlation coefficient (ICC) and/or coefficient of variation (CV) were included. The COSMIN checklist was used for the assessment of the methodological quality of the included studies.n = 1595) on test–retest reliability of 1RM assessment were found. All the studies were of moderate or excellent methodological quality. Test–retest ICCs ranged from 0.64 to 0.99 (median ICC = 0.97), where 92% of ICCs were ≥ 0.90, and 97% of ICCs were ≥ 0.80. The CVs ranged from 0.5 to 12.1% (median CV = 4.2%). ICCs were generally high (≥ 0.90), and most CVs were low (< 10%) for 1RM tests: (1) among those without and for those with some resistance training experience, (2) conducted with or without familiarization sessions, (3) with single-joint or multi-joint exercises, (4) for upper- and lower-body strength assessment, (5) among females and males, and (6) among young to middle-aged adults and among older adults. Most studies did not find systematic changes in test results between the trials.After reviewing 1024 search records, 32 studies , and sex or age of participants. Researchers and practitioners, therefore, can use the 1RM test as a reliable test of muscular strength. The 1RM test has good-to-excellent test–retest reliability.The reliability of the test seems to be high regardless of resistance training experience, number of familiarization sessions, exercise selection, part of the body assessed (upper vs. lower body), and sex or age of participants.Muscular strength can be defined as “the ability to exert a force on an external object or resistance” . Higher Resistance training is the most commonly used exercise intervention for increasing muscular strength . ResistaIn laboratory-based settings, muscular strength is most commonly assessed using isokinetic dynamometers . HoweverTest–retest reliability represents the consistency of results in a given test across repeated measurements , 20. RelThe test–retest reliability of the 1RM test varies significantly across different studies , 21–50. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for this systematic review . EnglishTo be included in the review, studies were required to meet the following criteria: (1) published in English and in a peer-reviewed journal, (2) investigated test–retest reliability of the 1RM test, and (3) presented ICC and/or CV values. As suggested by Koo and Li , ICC valTwo authors (JG and BL) of the review independently extracted the following data to an Excel spreadsheet: (1) details regarding the sample , (2) protocol used for the 1RM test , (3) ICC and/or CV values, and (4) any adverse events associated with the 1RM test. Coding files were checked between the authors, and all discrepancies were resolved through discussion and consensus.To assess the methodological quality of the included studies, Form B of the validated COSMIN checklist was used , which iThe searches through the databases yielded 1024 search results . Most of the studies were conducted among apparently healthy individuals with two studies examining the reliability of the 1RM test in clinical populations  or moderate (15 studies) methodological quality. The mean ± standard deviation values of the checklist were 9 ± 1 points (range = 8 to 11 points). The results of the quality assessment can be found in Table Test–retest reliability of 1RM assessment is summarized in Table Twenty-two studies included untrained individuals. ICCs for 1RM tests among untrained individuals ranged from 0.64 to 0.99 (median ICC = 0.97), where 92% of ICCs were ≥ 0.90, and 99% of ICCs were ≥ 0.80. The range of reported CVs was from 1 to 12.0% (median CV = 5.5%). Fourteen studies included individuals with some previous resistance training experience. ICCs for 1RM tests among individuals with previous resistance training experience ranged from 0.64 to 0.99 (median ICC = 0.98), where 93% of ICCs were ≥ 0.90, and 96% of ICCs were ≥ 0.80. The range of reported CVs was from 0.5 to 7.8% (median CV = 3.3%).Eighteen studies did not include a familiarization session. ICCs in these studies ranged from 0.64 to 0.99 (median ICC = 0.96), where 90% of ICCs were ≥ 0.90, and 96% of ICCs were ≥ 0.80. The range of reported CVs was from 1.0 to 9.0% (median CV = 5.3%). Fourteen studies included one or more familiarization sessions. In these studies, ICCs ranged from 0.64 to 0.99 (median ICC = 0.98), where 90% of ICCs were ≥ 0.90, and 93% of ICCs were ≥ 0.80. The range of reported CVs was from 0.5 to 12.1% (median CV = 3.8%).Seventeen studies used single-joint exercises. ICCs for 1RM tests using single-joint exercises ranged from 0.74 to 0.99 (median ICC = 0.97), where 93% of ICCs were ≥ 0.90, and 96% of ICCs were ≥ 0.80. The range of reported CVs was from 0.5 to 9.0% (median CV = 4.1%). Twenty-eight studies used multi-joint exercises. ICCs for 1RM tests using multi-joint exercises ranged from 0.64 to 0.99 (median ICC = 0.98), where 88% of ICCs were ≥ 0.90, and 92% of ICCs were ≥ 0.80. The range of reported CVs was from 1.0 to 12.1% (median CV = 4.3%).Twenty-one studies assessed upper-body strength. ICCs for 1RM tests of upper-body strength ranged from 0.64 to 0.99 (median ICC = 0.98), where 94% of ICCs were ≥ 0.90, and 96% of ICCs were ≥ 0.80. The range of reported CVs was from 1.0 to 7.9% (median CV = 4.1%). Twenty-eight studies assessed lower-body strength. ICCs for 1RM tests of lower-body strength ranged from 0.64 to 0.99 (median ICC = 0.97), where 86% of ICCs were ≥ 0.90, and 92% of ICCs were ≥ 0.80. The range of reported CVs was from 0.5 to 12.1% (median CV = 4.7%).Fifteen studies included female participants. ICCs for 1RM tests among females ranged from 0.64 to 0.99 (median ICC = 0.98), where 92% of all ICCs were ≥ 0.90, and 96% of ICCs were ≥ 0.80. The range of reported CVs was from 1.0 to 8.0% (median CV = 4.4%). Nineteen studies included male participants. ICCs for 1RM tests among males ranged from 0.64 to 0.99 (median ICC = 0.97), where 88% of all ICCs were ≥ 0.90, and 91% of ICCs were ≥ 0.80. The range of reported CVs was from 0.5 to 12.1% (median CV = 4.0%).Twelve studies included older adult participants. ICCs for 1RM tests among older adults ranged from 0.80 to 0.99 (median ICC = 0.97), where 93% of all ICCs were ≥ 0.90. The range of reported CVs was from 1.0 to 9.0% (median CV = 5.4%). Twenty-two studies included young to middle-aged adult participants. ICCs for 1RM tests among young and middle-aged adults ranged from 0.64 to 0.99 (median ICC = 0.98), where 91% of all ICCs were ≥ 0.90, and 97% of ICCs were ≥ 0.80. The range of reported CVs was from 0.5 to 12.1% (median CV = 3.5%).In 66% of the analyses that assessed potential systematic changes in 1RM test results between the repeated measurements, no significant changes were found. The remaining studies found higher 1RM values in the retest condition. For lower-body exercises, the reported increases in 1RM ranged from 1.1 to 17.5 kg (median = 5.5 kg). For upper-body exercises, the reported increases in 1RM ranged from 0.5 to 4.9 kg (median = 1.8 kg).The main finding of this systematic review is that the 1RM test generally has excellent test–retest reliability, regardless of the previous resistance training experience, sex, and age of the participants; whether or not the testing procedure includes familiarization sessions; whether the exercises are classified as single- or multi-joint movements; and whether the testing is conducted for upper- or lower-body musculature. This finding is based on 32 included studies that showed either excellent or moderate methodological quality.Research has established that the response to resistance exercise varies between resistance-trained and untrained individuals , 56. ForIn the Ploutz-Snyder and Giamis study , the autBesides training experience, variables such as exercise complexity have been suggested to play an impactful role in the reliability of the 1RM test . For exaEven though there are physiological differences between men and women, especially in muscle contractile properties, fiber type proportion, and perfusion , we founMost studies did not find systematic changes in results between the repeated measurements. In those that did, the observed changes were generally small. Their size was well below the average increases in strength commonly found in strength training interventions –67. ThisThe included studies were classified as having excellent or moderate methodological quality based on the COSMIN checklist. While 31 studies presented ICC values and thus received a point on item 11, one study presented only CV values , and sex or age of participants. No or only small systematic changes in 1RM are expected between repeated measurements. Researchers and practitioners can, therefore, use the 1RM test as a reliable test for assessing maximal dynamic muscular strength.


B. crudilactis and B. mongoliense genomes, two species from bovine milk origin. The ability of B. mongoliense to grow in media supplemented with whey or 3′SL was assessed. Next, the effects of cell-free spent media (CFSM) were tested against the virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.Human milk oligosaccharides (HMO) could promote the growth of bifidobacteria, improving young children’s health. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulent activity against intestinal pathogens. Bovine milk oligosaccharides (BMO), structurally similar to HMO, are found at high concentration in cow whey. This is particularly observed for 3′-sialyllactose (3′SL). This study focused on enzymes and transport systems involved in HMO/BMO metabolism contained in B. mongoliense presents a genome more sophisticated and more adapted to the digestion of BMO/HMO than B. crudilactis . In addition, HMO/BMO digestion involves genes encoding oligosaccharide transport systems found in B. mongoliense but not in B. crudilactis. B. mongoliense seemed able to grow on media supplemented with whey or 3′SL as main source of carbon . CFSM obtained from whey resulted in a significant under-expression of ler, fliC, luxS, stx1 and qseA genes  of E. coli O157:H7. CFSM from 3′SL resulted in a significant up-regulation of luxS  gene and a down-regulation of fliC  gene. CFSM obtained from whey resulted in significant up-regulations of sopD and hil genes  of S. Typhimurium, while CFSM obtained from 3′SL fermentation down-regulated hil and sopD genes .Due to the presence of genes encoding β-galactosidases, β-hexosaminidases, α-sialidases and α-fucosidases, B. mongoliense and its potential ability to metabolise 3′SL and whey, B. mongoliense seems well able to digest HMO/BMO. The exact nature of the metabolites contained in CFSM has to be identified still. These results suggest that BMO associated with B. mongoliense could be an interesting synbiotic formulation to maintain or restore intestinal health of young children.From enzymes and transporters highlighted in the genome of  Bacteroides spp. or Clostridium spp., other bacteria found in the microbiota . β-bonds , 12. In  β-bonds . In addi O157:H7 , 15, SalA 941256 , 17 and r jejuni .Because they have a similar structure, bovine milk oligosaccharides (BMO) share common properties with HMO. More than 60 BMO have been identified  and a siThe whey, a by-product issued from the dairy industry, is obtained after the casein precipitation . Its comB. longum subsp. infantis ATCC 15697 was improved up to 9.8-fold to HT-29 cells in presence of 3′SL and 6′sialyllactose (6’SL) [Lactobacillus acidophilus La-5 grown in medium supplemented with whey against enterohaemorrhagic E. coli (EHEC) infection in mice. Recently, Cooper et al. [B. animalis subsp. lactis CNCM I-3446 and whey permeate containing 3′SL had a strong bifidogenic effect on microbiota of children who were born to an HIV+ mother.The epithelial adhesion of e (6’SL)  and up te (6’SL) . Zeinhomr et al.  demonstrBifidobacterium mongoliense FR/49/f/2 and Bifidobacterium crudilactis FR/62/b/3, are able to tolerate presence of oxygen and acidity [Before reaching the colon to digest oligosaccharides, bifidobacteria must survive the stomach acidity, bile salts and pancreatic enzymatic activity. Most bifidobacteria do not support the presence of oxygen, making their production at industrial scale more challenging. Two bifidobacterial strains isolated from raw cow milk cheese,  acidity , 35. In  acidity . TherefoB. crudilactis LMG 23609 and B. mongoliense DSM 21395. The second aim of this work was to evaluate the potential growth of B. mongoliense FR/49/f/2 in culture media supplemented with whey or 3′SL and to assess the effects of filtered supernatants on virulence expression of E. coli O157:H7 and S. Typhimurium.The first aim of this study was to determine the enzymes and transport systems involved in BMO degradation using the complete genome of B. mongoliense DSM 21395, 57 genes encoding for glycoside hydrolases (GH) were identified  and the lowest count on MRS2–3′SL medium (6.7 ± 0.3 log cfu/mL). No growth was observed on MRS2 medium (6.0 ± 0.1 log cfu/mL)   . CFSM from MRS2-G medium tended to over-express the luxS gene (2.2) and two significant up-regulations of ler and stx1 genes  were observed  , while the significant increase of the stx1 gene expression was considered too low to be biologically meaningful . The details of the cycle threshold values corresponding to the effects of CFSM on E. coli O157:H7 genes expression are available in the Additional file After an incubation of 4 h, the CFSM had no negative impact on growth. The average OD of S. Typhimurium at 600 nm and after 4 h of exposure was 0.90 ± 0.04, while it was initially at 0.05 ± 0.02. CFSM obtained from MRS2 medium induced a significant up-regulation of the sopD gene    were noted for the CFSM from MRS2-G medium  was observed with CFSM obtained from MRS-3′SL medium , GH3 (glucosidases), GH13 (amylases), GH20 (β-hexosaminidases), GH33 , GH42  and GH95 (α-L-fucosidases). Those genes were found in the B. crudilactis LMG 23609 genome too, with exception of those encoding for GH20 and GH95 (involved in HMO or BMO digestion) and GH33, which could be considered an advanced genetic adaptation to the intestinal environment due to its involvement in degradation of both HMO/BMO and intestinal glycoconjugates such as mucin [B. mongoliense DSM21395 seems more efficient compared to B. crudilactis LMG 23609. Regarding B. crudilactis LMG 23609, eight potential genes are involved in milk sugar transport and most of them are involved in the transport of monosaccharides such as glucose and galactose. From the analysis of the TCDB families, B. mongoliense DSM21395 and B. crudilactis LMG 23609 could be able to transport LNB. However, LNB is a disaccharide associated with HMO rather than BMO [B. mongoliense DSM21395 or B. crudilactis LMG 23609.As explained previously, e origin  but B. mas mucin . In addithan BMO . Indeed,B. mongoliense could be able to digest most of the oligosaccharides found in BMO, except the sialic acid, which is supposed to stay in the external environment. In the same way, B. mongoliense could be able to metabolise 3′SL  using an extracellular α-sialidase. Then, the lactose  could be internalised using an ABC transporter and metabolised using a cytoplasmic β-galactosidase. According to this representation, the BMO or 3′SL degradation by B. crudilactis seems impossible without a previous cross-feeding step involving other bifidobacteria.According to the schematic representation of the BMO metabolism available in the Additional file B. mongoliense FR/49/f/2 showed good growth on media supplemented with either whey or glucose, whey contribution making no significant difference compared to glucose. In addition, whey is rich in lactose [B. mongoliense FR/49/f/2 grew on medium containing 3′SL as the main carbohydrate source, in comparison to the absence of growth observed on medium without any carbohydrate source. According to B. mongoliense DSM21395 genome analysis, this growth could result from the expression of the enzymes cleaving BMO or 3′SL bonds, such as α-sialidases, β-galactosidases and β-hexosaminidases. Genes involved in BMO degradation are also present in other bifidobacteria such as B. bifidum subsp. infantis and B. bifidum [ lactose , a carbo lactose , and in  lactose . Finally bifidum , 39, 40.C. jejuni [E. coli O157:H7 [S. Typhimurium [B. mongoliense FR/49/f/2 was able to decrease E. coli O157:H7 ATCC 43890 virulence. All the 5 tested virulence genes were significantly under-expressed . Furthermore, with CFSM obtained from medium fermented without any glucose, three genes were significantly down-regulated . In the study of Bondue et al. [B. bifidum BBA1 and B. crudilactis FR/62/B/3 presented a significant effect against E. coli O157:H7 ATCC 43890 virulence genes expression. Indeed, most of the virulence genes were down-regulated , except the fliC gene, which tended to be up-regulated. These results are different from what is observed in this study with B. mongoliense (significant down-regulation of fliC gene and significant up-regulation of luxS and stx1 genes). According to the genome of B. mongoliense, including many genes encoding enzymes from the GH family, metabolites produced in medium supplemented with 3′SL and fermented by B. mongoliense FR/49/f/2 could be different than those produced by B. crudilactis FR/62/B/3 and B. bifidum BBA1. Also, the down-regulation of the fliC gene while other virulence genes were up-regulated has already been demonstrated in other studies [E. coli or S. Typhimurium as described previously [CFSM issued from lactic acid bacteria or bifidobacteria contained metabolites decreasing virulence genes expression of . jejuni , E. coli O157:H7  and S. Thimurium . In our e et al. , CFSM ob studies –44. The eviously , 41.S. Typhimurium ATCC 14028 virulence gene expression was modulated, but not always in the same way as E. coli O157:H7 ATCC 43890. The CFSM from medium supplemented with glucose gave the same kind of regulation for both pathogens, and all the tested genes were over-expressed with S. Typhimurium . CFSM from media without any glucose or supplementation with whey down-regulated E. coli virulence genes, while these CFSMs induced mainly up-regulations of Salmonella virulence genes (hil and sopD). The only trend for down-regulation of Salmonella virulence genes was observed with CFSM supplemented with 3′SL (hil and sopD genes). The metabolites obtained from the fermentation of 3′SL by B. mongoliense could decrease pathogenicity of S. Typhimurium. This effect had already been observed with CFSM fermented by B. bifidum BBA1, where all the tested genes were under-expressed [After contact with the CFSM, xpressed .B. crudilactis FR/62/b/3 or B. mongoliense FR/49/f/2 has an antivirulent effect on E. coli O157:H7 and S. Typhimurium [B. crudilactis LMG 23609 genome but do not seem sufficient to easily metabolise the BMO. The enzymatic machinery proposed for BMO degradation identified in the B. mongoliense DSM21395 genome is more sophisticated with the putative presence of β-galactosidases, but also with α-sialidases and β-hexosaminidases. This highlights the putative capacity of B. mongoliense to metabolise the 3′SL and other complex BMO. The carbohydrate residues issuing from this degradation, such as sialic acid, glc, gal or GlcNac, could be involved in this antivirulent effect , and a size-exclusion chromatography in association with a mass spectrometry could contribute to further identifying them.Little is known about the exact nature of these metabolites. These bioactive molecules could originate from the degradation of proteins such as nisin or subpeptins similar to JM4-A and JM4-B, produced by lactic bacteria and having an antimicrobial effect , 46. Thehimurium  and thest Figure . The potB. mongoliense DSM21395 associated to the growth of B. mongoliense FR/49/f/2 on media supplemented with whey or 3′SL, B. mongoliense presents a genotype more adapted to the digestion of BMO compared to B. crudilactis. These two different kinds of metabolism observed for these two bifidobacteria species from bovine origin could have an impact on the nature of produced residual metabolites, which could differ in their influence on the virulence expression of pathogens. CFSM obtained from medium supplemented with whey and fermented by B. mongoliense FR/49/f/2 presented the most interesting effect by decreasing the virulence expression of the five tested genes of E. coli O157:H7 . For S. Typhimurium, the CFSM obtained after 3′SL fermentation presented the most interesting effect by decreasing 2 of the 3 tested genes (hil and sopD). According to this study, B. mongoliense FR/49/f/2 could be a potential probiotic, which associated to BMO, could maintain the young child’s gastrointestinal health through a synbiotic effect.In conclusion, according to the genome analysis of B. crudilactis LMG 23609 and B. mongoliense DSM 21395 are available on GenBank using accession number JHAL00000000 and JGZE00000000, respectively [The genomes of the two reference strains, ectively , 36, 47.B. mongoliense FR/49/f/2 and B. crudilactis FR/62/b/3 have been isolated from Saint-Marcellin, a raw cow milk cheese from the Vercors area (France). These strains as well as EHEC strain O157:H7 ATCC 43890 and S. enterica serovar Typhimurium strain ATCC 14028 were stored and grown following the experimental protocol described previously by Bondue et al. [e et al. .2HPO4, KH2PO4, NaCl, MnSO4.H2O, MgSO4.7H2O, FeSO4.7H2O and cysteine by Merck Laboratory . Sweet whey (12°D) was collected at the beginning of a curdling process in a Belgian cheese factory  and frozen at − 20 °C before further use. Whey was then sterilised using double filtration . The quantities of lactose and protein in MRS2-Wh medium were estimated to be around 25 g/L and 4 g/L, respectively [B. crudilactis FR/62/b/3 metabolism, were elaborated by Bondue et al. [B. mongoliense FR/49/f/2 was grown in three independent experiments on De Man, Rogosa, and Sharpe (MRS) medium  supplemented with cysteine-HCl (0.5 g/L) and mupirocin (0.08 g/L) under anaerobic conditions at 37 °C for 48 h. A maximum of two successive cultures have been carried out in MRS broth to reach 8 log/mL, prior to use. Next, the cultures were used to inoculate the four previously described media to reach 6 log/mL of bifidobacteria (1% v/v) . Bacterial growth was determined using viable plate counts after 48 h incubation. CFSM were obtained after two centrifugation steps at 5000 rpm  for 10 min. Supernatants were then sterilised by double filtration . Next, CFSM were freeze-dried  and rehydrated with sterile distilled water to obtain a 10-fold concentration. The same treatment was applied to non-fermented culture media (controls). To prevent an inhibition of pathogenic bacteria growth due to media acidification, the pH of rehydrated CFSM was adjusted to 7 using 1 M NaOH.Four media with different carbohydrate sources were used: a medium without any glucose MRS2), a reference medium with glucose (MRS2-G) , a mediu, a referectively . The 3′Sectively . The expe et al. . B. mongE. coli O157:H7 ATCC 43890 and S. Typhimurium ATCC 14028 were incubated overnight at 37 °C under agitation in LB and BHI broth, respectively. Volumes of 50 μL of homogenised cultures and 450 μL of each concentrated CFSM (fermented and unfermented) were then added to 4.5 mL of LB broth for E. coli and BHI broth for S. Typhimurium. Triplicate cultures were incubated at 37 °C for 4 h [E. coli O157:H7 and S. Typhimurium were grown in LB and BHI broth alone, respectively, as controls. Bacterial growth was determined by OD measurement at 600 nm.The applied method was fully described in the previous study of Bondue et al. . E. coli for 4 h  on a shaE. coli and to 50 ng/μL for S. Typhimurium. The RNA was then subjected to reverse transcription polymerase chain reaction (RT-PCR) using a high-capacity cDNA Reverse Transcription Kit . Synthesis of cDNA was performed in a Mastercycler Gradient Thermocycler  under the following conditions: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min and a cooling step at 4 °C. A no-RT control was made to confirm the absence of DNA contamination in each sample.Cells were then collected by centrifugation at 5000 rpm for 10 min at room temperature  and pellets were suspended in Tris-EDTA buffer containing 1% lysozyme . RNA was extracted using the RNeasy® Mini Kit  and DNA contamination was eliminated using the DNase I Recombinant RNase-free Kit . Samples were heated at 75 °C for 10 min in order to inactivate the DNase. The concentration of RNA was normalised to 100 ng/μL for E. coli O157:H7 ATCC 43890, the expression of ler, fliC, stx1, luxS, and qseA genes was determined using qPCR with gnd as a reference housekeeping gene [S. Typhimurium ATCC 14028, the virulence expressions of hilA, ssrB2 and sopD genes were assessed using gmk as a reference housekeeping gene [E. coli were: initial denaturation at 95 °C for 3 min; denaturation, annealing and elongation repeated 45 times: 95 °C for 15 s, 58 °C for 30 s and 72 °C for 45 s; melting curve program of 60–95 °C with a heating rate of 0.1 °C/s. The qPCR conditions for S. Typhimurium were: denaturation at 95 °C for 10 min; 40 cycles of amplification and quantification at 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s; melting curve program of 60–95 °C with a heating rate of 0.1 °C/s. The experiments were replicated three independent times and special attention was given to the controls to exclude any potential effect observed from the non-fermented media as described previously [Ct unfermented – Ct fermented / (reference gene Efficiency)Ct unfermented – Ct fermented, with Efficiency = 10(− 1/slope). The ratio obtained was considered with a calibrator ratio resulting from a control population (without modulation of gene expression). To determine a significant modulation of the ratio before and after contact with CFSM, a Wilcoxon test for paired samples was performed where p < 0.05 was considered as significant.To highlight the effects of filtrated supernatants on virulence gene expressions of ing gene , 51. Foring gene . Quantiting gene . The qPCeviously , 49. Theeviously : ratio =Additional file 1: Table S1. Cycle threshold values corresponding to the effects of tested CFSM on E. coli O157:H7 genes expression.Additional file 2: Table S2. Cycle threshold values corresponding to the effects of tested CFSM on S. Typhimurium genes expression.Additional file 3: Figure S3. Schematic representation of BMO and 3′SL metabolism by B. mongoliense and B. crudilactis.


Morus genus plants are mainly distributed in the temperate to tropical areas over the world and include 17 species and two subspecies. Due to their excellent pharmacological activity, security in food additives and high value in the national economy, Morus genus plants have drawn more and more attention in recent years. In the light of the references published over the last few decades, flavonoids, benzofurans, stilbenes, and Diels–Alder adducts have been reported to be the main bioactive constituents of Morus genus plants. This review summarizes the compounds with excellent bioactivities isolated from Morus genus plants as well as their structure–activity relationships (SARs), which might be useful for the further research and development of Morus genus plants.Morus genus plants as well as their structure–activity relationships (SARs) were summarized.The aromatic heterocycles with excellent bioactivities isolated from  Morus genus plants comprise flowering trees belonging to Moraceae family and are distributed in the temperate to tropical areas over the world. According to the webpage of https://www.theplantlist.org, the Morus genus comprises 17 species and two subspecies. In traditional Chinese medicine (TCM), the leaves, root, bark, stems, and fruits of M. alba are used for treatment of rheumatism, cough and inflammation, and the leaves and fruits of M. alba serve as foodstuff around the world. Recently, not only M. alba, but also other Morus genus plants’  chemical components and their biological activity have been evaluated  on α-glucosidase      -3′-methoxy-5′,6-dihydroxy-2-arylbenzofuran (31) were isolated from the root bark of M. alba var. tatarica. The IC50 values were 11.9 ± 1.3, 33.1 ± 1.9, 29.5 ± 3.4, 21.5 ± 0.5, 85.3 ± 2.7, 67.2 ± 3.2, 21.9 ± 1.7, 101.5 ± 6.5 and > 150 μM, respectively . The activity differences between them suggested that the substitution types of prenyl groups were important for α-glucosidase inhibitory effcts. Meanwhile, hydroxyl at C-3′ might be another functional group of benzofurans (29 vs 30). The abnormal behavior of 26 might be caused by the effect of the side chain  level in the blood. Pancreatic lipase (PL) is an enzyme that catalyzes triglyceride to fatty acids and glycerol. Inhibition of PL can decrease TG hydrolysis and reduce free fatty acid absorption from intestine to blood and finally lower blood TG level , 19. OrlMorus genus plants were reported to be PL inhibitors. Jeong et al. reported that the PL-inhibitory activities of both flavonols and flavanones from the leaves of M. alba would be decreased by the substitution of hydroxyl , the activities could also be reduced by glycosylation [quercetin (20) > quercetin-3-O-glucopyranoside (34)]  . Moreover, the prenyl group may also be an active unit in PL inhibition [moracins N (38) and C (39) > 35–37 and albafuran A (40)]   Fig. 3)M. alba, utyrate) . ThroughTyrosinase is an oxidase distributed in fungi, plants and animals widely. Generally, tyrosinase is known to be involved in melanin synthesis that gives skin, hair, and eyes color. In tumor cells, tyrosinase expression level is significantly up-regulated than in normal cells along with increase of tyrosinase activity. Tyrosinase inhibitors have attracted considerable attention in improving tumor immunity, such as imatinib, an antitumor drug against chronic myelogenous leukemia.Morus genus plants-enriched polyphenol had been used as a kind of non-toxic natural tyrosinase inhibitor to whiten skin   of flavonols  vs oxyresveratrol (62) with IC50 values at 6.9 ± 0.7 and 35.3 ± 3.7 μM, respectively; moracin C (37) vs moracin M (39) with IC50 values at 8.7 ± 0.3, 24.8 ± 0.0 μM, respectively] [For stilbenes and benzofurans from the wood of ctively]  Fig. 6)M. austraMorus alba has a long history of medicinal and edible usage in China. The relatively mature studies on its phytochemistry and pharmacology and clinical trials have led to enormous economic value [ic value .Morus genus plants have been summarized. Therefore, prenyl and hydroxyl substituted flavonoids compounds, benzofurans, stilbenes, and Diels–Alder adducts were found to possess significant bioactivities, which may provide some references for the seeking of effective substances with anti-diabetic, anti-obesity, neuroprotective and anti-cancer effects.On the basis of reports and reviews published, the SARs of α-glucosidase, lipase, tyrosinase, β-secretase, and acetylcholinesterase, and cytotoxicity of compounds obtained from


In the absence of echocardiography, identification of cardiomegaly via thoracic radiography is a necessary criterion for classification of disease severity in dogs with myxomatous mitral valve disease (MMVD).Modified‐vertebral left atrial size (M‐VLAS) facilitates objective radiographic assessment of the left atrium (LA) in 2 dimensions and identifies LA enlargement more accurately than existing methods.Sixty‐four dogs with various stages of MMVD and 6 control healthy dogs.Retrospective case–control study. Medical records were searched for dogs with varying severity of MMVD. Modified‐vertebral left atrial size, vertebral left atrial size (VLAS), vertebral heart size (VHS), and radiographic left atrial dimension (RLAD) were measured from thoracic radiographs and compared with echocardiographically derived measurements.r = 0.77, P < .001), VLAS , RLAD , and VHS . Receiver operating characteristic analyzes provided an area under the curve of 0.97  for M‐VLAS, which was superior to VHS  in identifying dogs with LA/Ao ≥1.6. A cut‐off value of ≥3.4 vertebrae using M‐VLAS provided 92.7% sensitivity and 93.1% specificity in predicting LA enlargement.Positive correlation to LA/Ao was identified for M‐VLAS (M‐VLAS, which is superior to VHS, offers an accurate and repeatable way to radiographically identify LA enlargement in dogs with MMVD. We hypothesize that M‐VLAS is superior to published methods in radiographic identification of LA enlargement given its additional dimension of LA measurement.2Electronic hospital records of dogs diagnosed with MMVD between 3 hospital sites of Veterinary Specialist Services  by the cardiology service from October 2017 to March 2020 were retrospectively reviewed. Dogs that were < 20 kg, with physical examination, 2 or 3 view thoracic radiographs , and echocardiogram performed within a 24‐hour period were included in the study. Echocardiograms were performed by either a board‐certified cardiologist (FEM/BJG) or a resident (CL) under their direct supervision. Echocardiographic measurements were taken from established 2‐dimensional (2D) views,A right lateral inspiratory thoracic radiograph from each dog was used for all radiographic measurements. Radiographic images were reviewed and measured using a commercial digital radiography viewing software . Vertebral heart size,Intraobserver agreement for all 4 radiographic measurements were determined with 1 investigator (CL) performing measurements on 3 separate occasions on 20 dogs randomly selected from the total population. Interobserver variability was determined by 3 investigators  completing all 4 measurement for the same 20 dogs. All investigators were blinded to the identity and disease staging of each dog.2.1P value <.05 was considered significant for all analyzes. Distribution of continuous variables was assessed for normality with D'agostino‐Pearson test and for normally distributed variables, values were expressed as the mean ± SD. One‐way analyzis of variance (ANOVA) with Tukey's multiple comparison test was used to compare age, bodyweight, M‐VLAS, VLAS, VHS, and RLAD between control and diseased dogs from various stages. Correlation of LA/Ao with M‐VLAS, VLAS, VHS, and RLAD was assessed using Pearson's correlation. Receiver operating characteristic (ROC) curves, and the area under the curves (AUC) with 95% confidence intervals (CI) using LA/Ao < or ≥ 1.6 as the classifiers were generated for M‐VLAS, VLAS, VHS, and RLAD. Comparison between the AUC of the 4 measurements was performed using Delong's method. Sensitivity and specificity for M‐VLAS, VLAS, VHS, and RLAD were determined with the Youden index to determine the optimal cut‐off value. Inter‐ and intraobserver variabilities were assessed for M‐VLAS, VLAS, VHS, and RLAD via intraclass correlation coefficient (ICC) estimates and their 95% CI based on a single rater, absolute agreement, with 2‐way random (interobserver) and mixed (intraobserver) effect. An ICC value of >0.9 was considered excellent, 0.75 to 0.9 was considered good, 0.5 to 0.75 was considered moderate, and poor if <0.5.Statistical analyzes were performed using commercial statistical software . A 3A total of 70 dogs were included into the study, comprising 64 dogs with MMVD  and 6 control healthy dogs. Within the stage B1 group , there were 5 mixed breeds, 4 Cavalier King Charles Spaniels (CKCS), 3 Maltese, 2 Chihuahuas, 2 Dachshunds, 2 Fox Terriers, 2 Poodles, 1 Miniature Schnauzer, and 1 Tibetan Spaniel. Stage B2 dogs  consisted of 6 mixed breeds, 6 CKCS, 2 Poodles, 2 Shih Tzus, 1 Beagle, 1 Chihuahua, 1 Dachshund, 1 Jack Russell Terrier, and 1 Maltese. Within the stage C group , there were 8 mixed breeds, 3 Chihuahuas, 3 Maltese, 2 CKCS, 2 Jack Russell Terriers, 1 Löwchen, 1 Silky Terrier and 1 Toy Poodle. Control dogs  included 1 Alaskan Klee Kai, 1 Fox Terrier, 1 Jack Russell Terrier, 1 Maltese, 1 mixed breed, and 1 Poodle. Table P < .001). No differences were observed between control and stage B1 dogs, nor between stage B2 and C dogs for any radiographic or echocardiographically‐derived value , VLAS , VHS , and RLAD . The ROC analyzes indicated that all radiographic methods were useful to identify dogs with LA enlargement (LA/Ao ≥1.6). Area under the curve for M‐VLAS, VLAS, VHS, and RLAD was 0.97 (95% CI 0.94‐1.00), 0.95 (95% CI 0.91‐1.00), 0.90 (95% CI 0.80‐0.96), and 0.93 (95% CI 0.85‐0.98), respectively. A cut‐off value of ≥3.4 vertebrae using the M‐VLAS method provided 92.7% sensitivity and 93.1% specificity (Youden index 0.86). Comparatively, a VLAS cut‐off value of ≥2.4 vertebrae provided 80.5% sensitivity and 96.6% specificity (Youden index 0.77). For VHS, a cut‐off value of ≥11.1 was 78.1% sensitive, and 96.6% specific (Youden index 0.75). Lastly, a cut‐off value of ≥1.7 using RLAD provided a sensitivity of 100% and specificity of 72.4% (Youden index 0.72). The AUC was significantly greater for M‐VLAS  compared to VHS  (P = .03). The AUC was similar for other radiographic methods.   Table .4The objectives of this study were to examine and compare different radiographic methods for identification of echocardiographically measured LA enlargement (LA/Ao ≥1.6) and to introduce a modified‐VLAS method that aimed to incorporate an additional dimension in measuring LA size on radiographs. The results of this study showed a positive correlation between M‐VLAS, VLAS, VHS and RLAD with LA/Ao in dogs with MMVD. Significantly higher M‐VLAS, VLAS, VHS, and RLAD were observed in stage B2 and C dogs than stage B1 and control healthy dogs. Optimal cut‐off values generated from ROC curves indicated that a M‐VLAS of ≥3.4 vertebrae offered 93% sensitivity and specificity in predicting significant LA enlargement as defined by a LA/Ao ≥1.6. Comparison of ROC curves indicated M‐VLAS was superior to VHS in identifying this LA enlargement.The original VLAS method aimed to quantify LA size radiographically based on a single‐dimensional measurement that roughly represented the transverse diameter of the LA body. Yet, LA functional anatomy and geometry is complex, with the left atrium described as a cylinder with an almost fixed head and distensible walls attached to a piston .r = 0.76, 95% CI 0.64‐0.85, P < .001). The optimal cut‐off value of ≥2.4 vertebrae derived from ROC analyzis in the current study to identify a LA/Ao ≥1.6 was close to the cut‐off value of ≥2.3 vertebrae originally proposed,The results of this study have reinforced the previously describedr = 0.77 vs. 0.67) and the AUC of M‐VLAS was greater than that of VHS . This is not a surprising result as VHS is a measurement of global cardiac size whereas M‐VLAS aims to more directly reflect LA size. Interestingly, neither VLAS nor RLAD which similarly aim to represent LA size demonstrated superiority over VHS by ROC analyzis. The additional dimension included in M‐VLAS measurement over the single axis measurement of VLAS and RLAD presumably facilitates a more comprehensive measure of LA size and the statistical distinction between M‐VLAS and VHS but not between VLAS, RLAD and VHS. Minimization of both false negatives and false positives is equally important when ascertaining the optimal measurement technique to radiographically identify cardiomegaly in dogs with MMVD. In the current study, M‐VLAS, with the cut‐off value of ≥3.4 vertebrae, was highly specific and sensitive (93%) at identification of LA enlargement (La/Ao ≥1.6), whereas VHS  provided high specificity (97%) at the expense of low sensitivity (78%).One of the most important findings of the current study is the superiority of M‐VLAS to identify LA enlargement compared to VHS. Figure  Both theMultiple publications have evaluated the radiographic method to objectively assess heart size by VHS since its introduction in 1995.26 or ≥ 1.7 (current study) as indicative of LA/Ao ≥1.6.The RLAD is proposed to assess LA enlargement in dogs with mitral regurgitation.There is no significant difference between VHS and VHS + VLAS in detection of LA enlargement (as defined by LA/Ao ≥1.6)The current study has several limitations. Firstly, because of the retrospective design sample sizes were small especially in the healthy group (n = 6). However, the aim of this study was not to differentiate between healthy and diseased dogs nor to establish normal reference ranges, but rather to assess the efficacy of objective radiographic measurements to identify LA enlargement (LA/Ao ≥1.6), which together with other cardiac criteria, is an indication for pimobendan therapy.Authors declare no off‐label use of antimicrobials.Authors declare no conflict of interest.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.


There is a clear need for innovation in anti‐tuberculosis drug development. The zebrafish larva is an attractive disease model in tuberculosis research. To translate pharmacological findings to higher vertebrates, including humans, the internal exposure of drugs needs to be quantified and linked to observed response.In zebrafish studies, drugs are usually dissolved in the external water, posing a challenge to quantify internal exposure. We developed experimental methods to quantify internal exposure, including nanoscale blood sampling, and to quantify the bacterial burden, using automated fluorescence imaging analysis, with isoniazid as the test compound. We used pharmacokinetic–pharmacodynamic modelling to quantify the exposure–response relationship responsible for the antibiotic response. To translate isoniazid response to humans, quantitative exposure–response relationships in zebrafish were linked to simulated concentration–time profiles in humans, and two quantitative translational factors on sensitivity to isoniazid and stage of infection were included.−1 internal concentration (minimum inhibitory concentration) leads to bacteriostasis of the mycobacterial infection in the zebrafish. The concentration–effect relationship was quantified, and based on that relationship and the translational factors, the isoniazid response was translated to humans, which correlated well with observed data.Blood concentration was only 20% of the external drug concentration. The bacterial burden increased exponentially, and an isoniazid dose corresponding to 15 mg·LThis proof of concept study confirmed the potential of zebrafish larvae as tuberculosis disease models in translational pharmacology and contributes to innovative anti‐tuberculosis drug development, which is very clearly needed. ADCacid–albumin–dextrose–catalaseCFUcolony‐forming unitsdpfdays post fertilizationdpidays post infectionFOCEfirst‐order conditional estimationGIgastrointestinalhpfhours post fertilizationLLOQlower limit of quantificationMGITmycobacteria growth indicator tubeMICminimum inhibitory concentrationMTPmultistate tuberculosis pharmacometricOADColeic acid–albumin–dextrose–catalasePVP40polyvinylpyrrolidone 40QCquality controlTBtuberculosisThe zebrafish is a well‐known tuberculosis disease model for qualitative efficacy screens in drug development.The information content and translational potential of qualitative assessments are low.Isoniazid exposure–response relationship was quantified in zebrafish larvae using integrated experimental and computational innovation.With pharmacokinetic–pharmacodynamic modelling, quantitative translation of pharmacological findings from zebrafish larvae to humans is confirmed.Adding zebrafish larvae to the translational drug development pipeline could expedite development of anti‐tuberculosis drugs.1Mycobacterium tuberculosis is becoming the deadliest pathogen on the planet  is the leading cause of death from infectious diseases in adults, and Phenotypic‐ and systems‐based drug development both integrate experimental and computational innovation and utilize whole‐organism studies, preferably in vertebrates, for quantitative translational purposes to open a new realm of possible discoveries that are easily overlooked when focusing on single cell‐type targets. High‐throughput experiments within whole vertebrates may improve efficiency and effectiveness of drug development and are possible with the zebrafish larva as model organism  is increasingly used in biomedical research, because of its many advantages, which include high fecundity, rapid development, transparency throughout the first period of life, easy genetic modification, and limited ethical constraints  (3.75–150 mg·L−1). The internal exposure is quantified in homogenates and blood samples of the larvae, and the bacterial burden is quantified by automated fluorescence image analysis, according to the design in Figure Here, for the first time, we present an integration of experimental and computational approaches in preclinical TB research, using zebrafish larvae infected with 22.1M. marinum strain E11 at 28 hours post fertilization (hpf) and kept at 28°C throughout the experiment. At 2 days post infection (dpi), the bacterial burden in zebrafish larvae was quantified by fluorescence microscopy, and waterborne treatment with isoniazid was commenced at external concentrations of 0, 3.75, 7.5, 15, 30, and 75 mg·L−1 corresponding to 0, 0.25, 0.5, 1, 2, and 5× MIC. Quantification of the bacterial burden was repeated within individual larva at 3 and 4 dpi to assess individual early bactericidal response. Internal isoniazid exposure was quantified by LC–MS/MS of whole zebrafish larval homogenate samples as well as in larval blood samples in a parallel experiment with uninfected larvae treated with external isoniazid concentrations of 7.5, 15, 30, 75, and 150 mg·L−1 corresponding to 0.5, 1, 2, 5, and 10× MIC. All replicates are biological replicates; technical replicates were not obtained. Figure Zebrafish embryos of the VUmc wild‐type strain were dechorionated and infected with 2.2−1, volume 10 L, 120 × 220 × 490 mm; Fleuren & Nooijen BV, Nederweert, The Netherlands) with circulating water . Adult zebrafish were fed twice daily with artemia or feed particles . JUMO ACQUIS touch S  was used to control water quality.Zebrafish were maintained and handled following international consensus protocols  at 28°C. For the duration of treatment, larvae were kept in embryo medium with isoniazid at 28°C until imaging or sampling.Adult zebrafish were set‐up for breeding overnight, and in the morning at lights on, separators between males and females were removed, and fertilized eggs were collected within 30 min of fertilization. Eggs, embryos, and larvae were kept in embryo medium (60 μg·ml2.3−1). Experiments were performed on different days per dose, and the experimentalist was unblinded to the external concentration when blood samples or homogenates were obtained. As no outcome measurements were acquired at this stage, this was not expected to result in measurement bias.Internal exposure of isoniazid in zebrafish larvae was quantified as drug amounts in larval homogenates or as drug concentrations in blood samples after waterborne treatment with isoniazid concentrations at external concentrations of 0.5, 1, 2, 5, and 10× MIC , which was repeated to check for inter‐day variability, thus resulting in n = 6 replicates. Zebrafish larvae were washed 4 times with 20/80 methanol/water (v/v) using Netwell inserts  and transferred to Safe‐Lock tubes . Excess volume was removed, and 50 μl of 200 ng·ml−1 isoniazid‐D4 internal standard was added after which the larvae were snap frozen in liquid nitrogen and stored at −80°C until quantification.Homogenate samples of pooled samples with five larvae at time points 0.25, 0.5, 1, 2, 3, 6, 8, 9, 18, 20, 22, 24, 26, 32, 42, 44, 46, 48, and 50 h after start of treatment were obtained at n = 4) were taken after 48 h of treatment at the highest external concentration of 150 mg·L−1 to ascertain quantifiable levels, using a previously published method  and pooled into a 0.5‐ml tube (Eppendorf Nederland B.V.). Blood samples were kept at −80°C until quantification. Table Blood samples .g; 90% was transferred to a new Safe‐Lock tube and evaporated until dryness in a Labconco vacuum centrifuge . The residue was reconstituted into 200 μl methanol, centrifuged for 10 min at 20,000× g, and the supernatant was transferred to an LC–MS/MS vial with a glass insert for 5 μl injection into the LC–MS/MS. Quality control (QC) and calibration curve samples were prepared by adding known amounts to blank homogenate samples. The QC samples were prepared at the levels of 4, 125, and 225 ng·ml−1. The calibration curve samples were prepared at the levels of 0, 2, 5, 10, 25, 50, 100, 150, and 250 ng·ml−1.Samples of whole zebrafish larva were thawed, and 100 μl methanol and 100 μl of 0.5 mm zirconium oxide bullets  were added. The samples were homogenized using a Bullet Blender (NextAdvance) for at least two rounds of 5 min at speed 5. Extraction and derivatization were performed by adding 800 μl acetonitrile, 100 μl 1% cinnamaldehyde in methanol, and 100 μl formic acid and shaking for 20 min at 650 r.p.m. . Samples were centrifuged for 10 min at 20,000× −1 internal standard solution was added. Extraction and derivatization were performed by adding 200 μl acetonitrile, 50 μl methanol, 25 μl 1% cinnamaldehyde in methanol, and 25 μl formic acid, and samples were shaken for 20 min at 650 r.p.m. Samples were evaporated until dry, reconstituted in 10 μl methanol, and centrifuged for 10 min at 20,000× g, after which they were transferred to an LC–MS/MS vial with a glass insert upon 5 μl injection into the LC–MS/MS. An academic calibration curve, in methanol without biological matrix, was prepared with concentrations 0, 2, 3, 5, 8, 10, 25, 50, 100, 150, and 250 ng·ml−1.Blood samples were thawed and briefly centrifuged to concentrate the small sample into the bottom of the tube. A 2.5 μl of 200 ng·mlQuantification of isoniazid was performed on an ultra HPLC system  with a triple quadrupole MS detector . Electron spray ionization in positive modes was used to obtain derivative ions.−1 on a Luna Omega Polar C18 1.7 μm 100 × 2.1 mm column with a 5‐mm guard column with the same packing material . The temperature of the column was maintained at 40°C. Gradient elution was performed with two ultra HPLC pumps using methanol/water mixtures with 0.01% formic acid. The gradient started at the time of injection and increased from a 49/51 methanol/water v/v ratio to a 72/28 methanol/water v/v ratio within 4 min. The column was flushed with 95/5 methanol/water v/v ratio starting at 4.1 min for 2.4 min, after which the system was equilibrated to initial conditions.Chromatography was performed at a flow of 0.4 ml·min+ = 252.1 m/z) and the isoniazid‐D4‐adduct (MH+ = 256.1 m/z). For the isoniazid derivative, the fragments were 79.01 and 121.01 m/z, and for the internal standard derivative, the fragments were 83.10 and 124.96 m/z. The sheath gas pressure was 40 psi, auxiliary gas pressure was 15 psi, S‐lens RF amplitude was 63 V, and capillary pressure was 1.190 mTorr. Detection limit was 0.5 ng·ml−1, and lower limit of quantification (LLOQ) was 1.75 ng·ml−1. The LC–MS/MS method was validated according to the US Food and Drug Administration guidelines , and the colony was suspended in 7H9 supplemented with 10% acid‐albumin‐dextrose‐catalase (ADC) and cultured overnight at 28°C. The OD at 600 nm (OD600) of bacteria was measured the next day . The logarithmic phase bacteria were harvested and washed 3 times with sterile PBS. The infection inoculum was resuspended to a target concentration of 200 CFU·nl−1 in 2% polyvinylpyrrolidone‐40 solution (PVP40). The MIC of the used strain had been determined to be 15 mg·L−1, in line with reported values , 10 min prior to injection and injected with 1 nl of 200 CFU·nl−1M. marinum E11 using the microinjection system , into the caudal vein at the blood island. After injection, the embryos were kept at 28°C. To quantify the established infection at 2 dpi, a Fluorescence Stereo Microscope  equipped with the digital camera  was utilized for fluorescence imaging, after which the larvae were transferred to 96‐well plates  with isoniazid solutions of 0, 3.75, 7.5, 15, 30, or 75 mg·L−1 in embryo medium. Group size was designed at n = 20, and final group size taking into account experimental loss of larvae was n = 17, n = 20, n = 17, n = 19, n = 18, and n = 19 for 0, 3.75, 7.5, 15, 30, or 75 mg·L−1 isoniazid, respectively. At 3 and 4 dpi, fluorescence imaging of individual larvae was repeated, after which they were transferred to a new 9‐well plate with fresh isoniazid treatment solution (3 dpi) or sacrificed by tricaine overdose (4 dpi).The injection of zebrafish larvae with The experimenter was unblinded to the dose, but the potential of measurement bias was limited as image analysis was automated. Automated image analysis was performed as reported before   elimination was estimated. Concentration in the treatment medium was assumed to be constant, as supported by measurements of external drug concentrations Figure .We expected age to be a covariate (predictor) on the absorption and elimination rate constants, but because the effects of age on absorption and elimination are indistinguishable at steady state, we have estimated a net effect of age as covariate for a net increase on absorption only, which reflects the relative increase  in absorption rate constant, compared to the increase in elimination rate constant. For this, we tested a linear, power, or exponential relationship. In addition, a discrete increase in absorption rate constant between 3 and 4 dpf to reflect the effects of the opening of the gastrointestinal (GI) tract , kg the growth rate (h−1), and Bmax the maximum capacity of the system (log10 fluorescence). Because no ceiling of bacterial growth was observed in the data, the maximum capacity of the system could not be estimated for the Gompertz or logistic growth function. Growth and decay could not be estimated separately as this was mathematically not identifiable, given the data. Therefore, a net effect was estimated for growth, which means a negative growth represents a net decay or kill of bacteria.The pharmacodynamic component of the model was fitted through logEquation , or logiEquation  growth fCINH is the concentration of isoniazid (ng·μl−1 or mg·L−1), AINH is the amount of isoniazid (ng) in the homogenate samples, and Vd is the scaled volume of distribution (μl) on each day.Based on the homogenate and blood sample data at 5 dpf, the distribution volume at this age was estimated. This distribution volume was subsequently scaled to 3 and 4 dpf based on total larval volume quantified previously −1], Emax the maximal response (−), EC50 the concentration of isoniazid responsible for 50% of the maximal response (mg·L−1), and γ the Hill exponent (−).For the exposure–response relationship (EFF), linear Equation , Emax  variability in the bacterial burden.Biological  variability was tested on the estimate of the inoculum, as well as on the parameters of the exposure–response relationship and reported as coefficient of variation. A proportional error model, parameterized as an additive error on the logP < 0.05 between models with a single degree of freedom difference, assuming a χ2 distribution. Precision of the estimates of structural parameters was considered to be acceptable when relative standard errors of these estimates remained below 50%, which is a second means of assessing that the model is supported by the data. The physiological plausibility of parameter estimates and visual assessment of goodness‐of‐fit plots . The exponential growth rate of the bacterial burden was assumed to be similar, irrespective of measurement of bacterial burden by fluorescence (zebrafish) or CFU·ml−1 (humans).Second, the simulated concentrations for the 1,000 individuals per dose group were linked to the exposure–response relationship for Third, the obtained isoniazid response was quantitatively compared to published bacterial burden data in humans . Nanopure water was used from a PURELAB water purification system  unless otherwise stated. ULC/MS‐grade methanol as well as ULC/MS‐grade acetonitrile, LC/MS‐grade water, and formic acid was acquired from Biosolve B.V. . Difco Middlebrook 7H10 agar and 7H9 medium, OADC, and ADC were acquired from Becton Dickinson and Company .33.1−1) was quantified. Figure Internal exposure of isoniazid in zebrafish larvae after constant treatment between 3–5 dpf with increasing doses—defined here as external isoniazid concentration in the treatment medium—of 0.5, 1, 2, 5, and 10× MIC  with 150 mg·L3.2M. marinum was quantified through fluorescence imaging with repeated measurements of each individual larva. Doses ranging from 0.25 to 5× MIC were chosen based on a feasibility study and on the fluorescence detection limit. Figure The bacterial burden of 3.3To quantify the exposure–response relationship, a sequential modelling approach was performed. First, the internal exposure over time was quantified in the pharmacokinetic component of the model, after which this was linked to the isoniazid response in the final pharmacokinetic–pharmacodynamic model Figure .ka) in two ways: first, in an exponential relationship per hpf, and second, based on knowledge on the physiology of the GI tract, which opens between 3 and 4 dpf  compared to an exponential relationship, and a power relationship was statistically similar (P > 0.1) but resulted in worse precision of the parameter estimates.A one‐compartment model with first‐order absorption and first‐order elimination best described the data on internal exposure. Because the larvae are still developing, both absorption and elimination are expected to increase with age , and as the opening of the GI tract was physiologically expected to affect absorption, the relationship was retained despite the slight imprecision of the obtained estimate. The precision of the remaining model parameters confirmed that the obtained model and parameter values are supported by the data. Goodness‐of‐fit plots further confirm an unbiased fit of the data by the model , with only the relative standard error of l Figure . A visuaThe data of bacterial burden did not support separate estimation of growth and decay; therefore, only the net effect of these two processes was estimated in the model, which was found to be best described with an exponential growth model. In case of bacterial kill exceeding bacterial growth with sufficient isoniazid response, this net growth rate would become negative. A linear exposure–response relationship for inhibition of bacterial growth fitted the data best. The biological variability between larvae was quantified by inclusion of inter‐individual variability on the inoculum (coefficient of variation 204%) and slope of the drug response (coefficient of variation 50.5%). The experimental variability was very reasonable with a coefficient of variation of 36.3%. The predicted isoniazid response in zebrafish larvae is shown in Figure 3.4M. tuberculosis in humans to be similar to the isoniazid response of its close relative M. marinum in the larvae. Two translational factors were taken into account  model has been developed previously for 10 CFU·ml−1·day−1 after 2 days of treatment, has been reported in humans before  obtained from simulated concentration–time profiles in humans and the exposure–response relationship quantified in zebrafish larvae here. The median simulated decline in the bacterial burden of 0.7–0.9 logIn conclusion, we have developed a new experimental and computational approach to translate the pharmacokinetic–pharmacodynamic relationship of the early bactericidal response to an antibiotic in a zebrafish model of TB to humans. We propose that this approach is used in the search for novel TB treatments.R.C.v.W., U.S.H.S., H.P.S., P.H.v.d.G., and E.H.J.K. contributed to the conceptualization of this study; R.C.v.W., W.H., and S.M.D. contributed to the investigation; J.L., R.B., D.J.v.d.B., and F.J.V. contributed to the methodology; R.C.v.W. and E.H.J.K. contributed to the formal analysis and writing of the original draft; and W.H., S.M.D., J.L.,. R.B., D.J.v.d.B., F.J.V., U.S.H.S., H.P.S., and P.H.v.d.G. contributed to the writing (review and editing) of the paper.The authors declare no conflicts of interest.BJP guidelines for Design and Analysis, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the Figure S1. Goodness‐of‐fit plots for the pharmacokinetic component of the final pharmacokinetic‐pharmacodynamic model in zebrafish larvae.M. marinumFigure S2. Model‐based individual prediction of the bacterial burden after isoniazid treatment in zebrafish larvae infected with Figure S3. Goodness‐of‐fit plots for the pharmacodynamic component of the final pharmacokinetic‐pharmacodynamic model in zebrafish larvae.Figure S4. Stability of waterborne isoniazid over treatment period.Table S1. Overview of the blood sampling experiment, reporting the number of larvae sampled per timepoint, the total blood volume, and the isoniazid concentration.M. marinumModel code: Final pharmacokinetic‐pharmacodynamic model in zebrafish infected with Model code: Translation to human isoniazid responseClick here for additional data file.


Babesiosoma Jakowska and Nigrelli, 1956 and Dactylosoma Labbé, 1894. These blood protozoa occur in peripheral blood of lower vertebrates, and are commonly reported parasitising amphibians. The present study describes Dactylosoma piperis n. sp. from the pepper frog Leptodactylus labyrinthicus  (Anura: Leptodactylidae), collected in 2018 at the municipality of Araguaiana, Mato Grosso State, Brazil, based on morphology of intra-erythrocytic trophozoite, primary and secondary merogonic stages and a molecular analysis . Dactylosoma piperis n. sp. forms a well-supported clade with other Dactylosomatidae. This is the first molecular characterization of a species of Dactylosoma from a Brazilian anuran.The Dactylosomatidae Jakowska and Negrelli, 1955 are one of four families belonging to adeleorinid coccidia and comprise the genera  Dactylosoma Labbé, 1894 and Babesiosoma Jakowska and Nigrelli, 1956 .Gene sequence: 18S rRNA gene sequence deposited in GenBank under accession number MW264134.Note: The authors of the new taxon are different from the authors of this paper; Article 50.1 and Recommendation 50A of the International Code of Zoological Nomenclature [nclature .Dactylosoma observed were trophozoites, early stage meronts, meronts and merozoites from the primary merogony. For secondary merogony, it was possible to identify early stage meronts, meronts and merozoites. In addition, the early stage meronts and the mature meronts varied in morphology including the typical hand-like (dactylate shape), the quadrangular, the fan-like and circular shapes. Typically, primary merogony of species of Dactylosoma produces up to 16 merozoites and secondary merogony up to eight merozoites. However, in the present study, during primary merogony, meronts were observed producing up to ten chromatin divisions of the nuclei and during secondary merogony, meronts were observed producing up to eight chromatin divisions .phozoite : ElongatYoung primary meronts , meronts : Ovoid tPrimary meronts . meronts –1D: LargPrimary meronts with merozoites (2 ± 0.5 μm2 (n = 3); multinucleate with ovoid dense chromatin positioned on one side of the parasite, usually displacing erythrocyte nuclei, chromatin staining dark purple or pinkish; merozoites measurements 7.45 μm ± 0.25 μm in length and 2.90 μm ± 0.25 μm in width (n = 30).rozoites –1F: LargYoung secondary meront (2 ± 0.2 μm2 in area (n = 3).y meront –1H: ElonSecondary meronts (2 in area (n = 2); multinucleate with between five and eight nuclei located peripherally with dense chromatin staining in deep magenta. meronts –1J: DactSecondary meronts with merozoites (2 (n = 2). Merozoites measured 6.2 μm ± 0.2 μm long and 1.5 μm ± 0.9 μm wide (n = 12). Fan-like shape meront (2 (n = 1). Merozoites measured 5.88 μm ± 0.2 μm long and 1.3 μm ± 0.9 μm wide (n = 8).rozoites –1L: Morprozoites : Multinue meront : MultinuDactylosoma piperis n. sp. is characterised by its elongated and unique trophozoites, with one side rounded and the other tapered; the morphological variation of early stage meronts to mature meronts ranging between dactylate, fan-like, quadrangular and circular shapes, and the number of merozoites produced in primary merogony (up to 10) and secondary merogony (up to eight).Dactylosoma from anuran hosts, namely D. kermiti, D. ranarum, D. sylvatica, and D. taiwanensis based on several developmental characteristics, such as the number of nuclear chromatin divisions present in primary and secondary merogony, unique trophozoite morphology and developmental stage morphometrics.This species can be distinguished from all currently recognised species of D. piperis n. sp. differs from D. ranarum (the first described species in the Dactylosomatidae), in the number of chromatin divisions of up to six nuclei during secondary merogony and trophozoite morphology being slender and smaller with both ends rounded. Nevertheless, certain characteristics observed, such as meronts with merozoites arranged in fan-like fashion or quadrangular mass, and the two types of schizogony (primary and secondary), are typical of dactylosomatid parasites. The first type producing larger meronts with nuclei located peripherally and vacuoles present during merozoite formation, and the second type producing smaller meronts, with chromatin division of nuclei more condensed and staining dark-purple with fewer merozoites produced.In comparison, D. kermiti, primary merogony is characterised by up to 14 chromatin divisions and second merogony by up to six chromatin divisions, as compared to D. piperis n. sp., with up to 10 and up to 8 chromatin divisions observed in primary and secondary merogony, respectively. In addition, the trophozoites of D. kermiti are smaller and slender, and elongated to oval in shape with vacuoles present, measuring 6.7 μm ± 2.2 μm long and 3.5 μm ± 1.2 μm wide, which differs from the trophozoite size and shape of D. piperis n. sp. Also, the morphometric values of primary meronts and merozoites, and secondary meronts and merozoites from this study were larger as compared to D. kermiti.For D. sylvatica and D. piperis n. sp. differ in morphology and size, with trophozoites of D. sylvatica measuring lager (7.0–8.5 μm × 6.3–7.6 μm), with an amoeboid shape, circular nuclei and alveolar cytoplasm without inclusions. Furthermore, meronts of D. sylvatica produce only up to eight merozoites, in the first and the second merogony, as compared to up to ten in the first merogony and eight in the second merogony of D. piperis n. sp.In comparison, the developmental stages between D. taiwanensis and D. piperis n. sp., both species present similar trophozoite morphology, with trophozoites of D. taiwanensis measuring 3.9 μm wide and 7.3 μm long; distinguished morphology can be observed, with elongate or ovoid vacuolated form with equal rounded ends, compared to elongated with one end rounded and the other tapered from D. piperis n. sp. From secondary merogony, mature fan-like, quadrilateral-shape and hand-like meronts were observed with between four and eight nuclei, differing from the mature meronts of D. piperis n. sp. with between six and eight nuclei.With regard to Haemogregarina forming a monophyly sister to a large clade consisting of isolates from species of Hepatozoon, Hemolivia, and Karyolysus. All species of Dactylosoma clustered together as a sister group to the Haemogregarinidae clade  available from GenBank . The BI ae clade .Dactylosoma piperis n. sp. (MW264134) is well nested within the Dactylosomatidae clade, forming a sister taxon to D. kermiti (MN879398/MN879392). Moreover, the genetic distances of the isolate from this study and dactylosomatid sequences available on GenBank showed interspecific divergence of 0.63% with D. kermiti (MN879398/MN879392) and 1.90% with D. ranarum (HQ224957/HQ224958), and the pair-wise distance varied from 0.005 to 0.009 (452 nt) ((452 nt) .Amphibians are experiencing large-scale declines in species diversity. According to the IUCN Global Amphibian Assessment over the past decade, a third of the estimated amphibian species have declined. The major contributors to amphibian’s species declines are environmental changes, fragmentation, and loss of habitat , 30. In Costa and Bérnils  reportedL. labyrinthicus was infected by a species of Dactylosoma. This anuran is widely distributed throughout South America [L. labyrinthicus occurs mainly near wetlands and has been recorded in open habitats throughout the Cerrado, Caatinga regions, and in central Amazonia [Leptodactylus group [The  America , 36, 37.Amazonia , 52, 57.us group  and oppous group , 78, 81.us group .L. labyrinthicus, a new species of Dactylosoma, Dactylosoma piperis n. sp. is described, with parasitaemia of 0.2%. In a recent study, Netherlands et al. [D. kermiti infecting anurans in South Africa, with parasitaemia varying between host species and individuals. In the host Ptychadena anchietae , parasitaemia varied from 2% to 5.7%, and in the host Sclerophrys gutturalis , parasitaemia averaged 0.2%, similar to the current study’s findings.In the present study from the blood smears of s et al.  describeHaemogregarina from 100 anurans screened. Intra-erythrocytic gamonts infecting the blood smears of one Rhinella crucifer  (syn. Bufo crucifer) from Rio de Janeiro State, Brazil, were found with parasitaemia 0.5%.In Brazil, studies of haemogregarine prevalence and parasitaemia from anurans are scarce. Da Sousa and Filho  reportedRhinella diptycha  (syn. Bufo paracnemis) collected at João Pessoa City, Paraíba State, Brazil. However, gametocyte morphology was similar to that of the genus Hemolivia.In another study by Kattar , from 10Leptodactylus chaquensis Cei, 1950, L. podicipinus Cope, 1862 and Phyllomedusa hypocondrialis Daubin, 1800, from Mato Grosso do Sul State and São Paulo State.Using microscopy screening of blood smears, Leal et al.  reportedDactylosoma, Da Costa and Pereira [Dactylosoma infecting L. latrans  (syn. L. ocellatus) from Rio de Janeiro State with low prevalence reported only in the fall and winter season (1964–1971); however, no morphometric data are available for these observations. The only developmental stages reported were meronts conforming to secondary merogonic early meronts with nuclei located at the rounded periphery of the parasite, and a fan-like shaped meront with four nuclei.Regarding species of  Pereira  observedDactylosoma have a wide distribution, infecting a variety of hosts [D. piperis n. sp., is a previously described species from a different continent, with different biomes, ecosystems and also different vertebrate hosts and possible vectors. All these data support the description of D. piperis n. sp. as a new species with the aid of morphological and molecular analysis.Species of of hosts , 53, 60.of hosts , suggestHepatozoon were polyphyletic, with species isolated from large mammals forming a well-supported clade sister to the Karyolysidae clade comprising species of Karyolysus, with species of Hepatozoon isolated from amphibians, reptiles and rodents forming a well-supported clade sister to the Karyolysidae clade comprising species of Hemolivia. The Haemogregarinidae clade formed a sister clade to the large monophyly comprising the Hepatozoidae and Karyolysidae clades. The Dactylosomatidae clade was found to be a well-supported monophyletic group sister to the Haemogregarinidae clade, these findings are similar to those reported by Netherlands et al. [With regard to the molecular analysis, the phylogenetic relationships between different haemogregarines  and the isolate from the present study showed the forming of several well-supported clades. Species of s et al. Babesiosoma stableri (HQ224961); Dactylosoma sp. (MN879399) and D. ranarum (HQ224957/HQ224958) formed a monophylum; and D. kermiti (MN879398/MN879392) and D. piperis n. sp. (MW264134) nested within the polytomy. Despite the low interspecific divergence (p-distance 0.005–0.009) between dactylosomatid species, the 18S gene distinguished D. piperis n. sp. as a separate species. Thus, although the 18S rRNA gene is a conservative marker, it has provided stability between closely related genera and species within Adeleorina [In addition, the Dactylosomatidae clade formed a polytomy with ri HQ2249; Dactyloeleorina , 70.Hepatozoon, the genus Bartazoon Karadjian, Chavatte and Landau, 2015, was proposed to replace the Hepatozoon genus from species transmitted exclusively by haematophagous insects, with the aim of resolving the polyphyletic placement of the genus Hepatozoon [Hepatozoon polyphyly still remain unsolved even with the use of the proposed genus Bartazoon.Moreover, in an attempt to resolve the polyphyletic genus patozoon . Howeverpatozoon  considerBartazoon based on the congenic and phylogenetic relationship of Hepatozoon griseisciuri and the type species of the genus Hepatozoon as described by Miller [Hepatozoon spp., showed significant pairwise differences observed between 18S rDNA and mitochondrial genome sequences; the sequences observed in their study support the idea of superiority of COI sequences on nuclear genes to describe species, and the mitochondrial genomes sequenced to date display staggering diversity. The adeleorinid coccidian will require additional sequence data from mitochondrial genomes to better understand the taxonomy and phylogenetic classifications, and the authors suggested that the genus Hepatozoon is likely distributed into multiple genera that have yet to be defined.Furthermore, Léveillé et al , shows ty Miller . Léveilly Miller  reportinHepatozoon spp., covering different groups of animals and transmission pathways, considered valid so far.Notwithstanding, until the scientific community has complete knowledge about the transmission structure and life cycle of haemogregarines, as well as the real phylogenetic and genomic diversity, the addition of a new genus to the group is precipitated. Thus, the phylogenetic analysis from this study was based on Léveillé et al.  and MaiaD. piperis n. sp., should be attempted to gain a better understanding of the ecology of this parasite.The importance of using techniques to correctly identify and describe a new species is emphasized in this manuscript. However, regarding the molecular technique, future studies should include using variable markers, such as a mitochondrial gene, to increase the phylogenetic resolution and systematic position on dactylosomatid parasites. Furthermore, studies including a great variety of Brazilian anuran species from different localities should be done with the aim of increasing the biodiversity and prevalence knowledge of dactylosomatid species. Also, studies focusing on life-cycle experimental work, testing possible vectors in the transmission Dactylosoma parasitising Brazilian anurans.This study provides the first report with molecular characterisation of a species of The authors declare that they have no conflict of interest.R.J.S. is supported by FAPESP (2016/50377-1), CNPq (309125/2017-0), and CNPq-PROTAX (440496/2015-2). L.P.U is supported by FAPESP . L.H.O is supported by FAPESP (2018/09623-4).