FANCA in 29 North African FA patients from Tunisia, Libya, and Algeria. Cytogenetic tests revealed high rates of spontaneous chromosome breakages for all patients except two of them. FANCA molecular analysis was performed using three different molecular approaches which allowed us to identify causal mutations as homozygous or compound heterozygous forms. It included a nonsense mutation , one reported missense mutation , a novel missense variant , and the FANCA most common reported mutation . Furthermore, three founder mutations were identified in 86.7% of the 22 Tunisian patients: (1) a deletion of exon 15, in 36.4% patients (8/22); (2), a deletion of exons 4 and 5 in 23% (5/22) and (3) an intronic mutation c.2222 + 166G > A, in 27.3% (6/22). Despite the relatively small number of patients studied, our results depict the mutational landscape of FA among NA populations and it should be taken into consideration for appropriate genetic counseling.Populations in North Africa (NA) are characterized by a high rate of consanguinity. Consequently, the proportion of founder mutations might be higher than expected and could be a major cause for the high prevalence of recessive genetic disorders like Fanconi anemia (FA). We report clinical, cytogenetic, and molecular characterization of  Fanconi Anemia (FA) is a rare, inherited disorder clinically characterized by various congenital abnormalities and a predisposition to develop malignancies, especially acute myeloid leukemia, and other cancers . The proFANCB which is X-linked  for confirmation of FA based on the standard test involving MMC-induced chromosomal breakage analysis. The cytogenetic and molecular status of 30 families with 46 patients have been previously described . Since FThis study was conducted according to the principles of the Declaration of Helsinki and was approved by the biomedical ethics committee (2017/14/I/LR11IPT05/V0) of Pasteur Institute. Informed consent of the legal patients’ representatives were obtained. Peripheral blood samples were collected from the 29 FA patients, their parents, and their siblings when available.Genomic DNA was extracted using the standard salting-out method  or the QClinical examination and familial information details were recorded. Clinical data were obtained based on history, physical examinations, and reviewing of medical records.FANCA gene (LRG_495) which is a common founder mutation in Southern Tunisia  for  patients. PCR products were analyzed using ABI PRISM 3130 Genetic analyzer (Applied Biosystems).Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large deletions or duplications within the FANCA gene (D16S3026‐ D16S3121‐ D16S3407) as previously reported  patients, available family members were genotyped with three microsatellites flanking the reported .HS (high sensitivity) workflow as a target enrichment method. Amplicon libraries were prepared from patients’ genomic DNA using the HaloPlexHS PCR target enrichment system dedicated to Ion Torrent PGM according to the manufacturer’s recommendations. Massively parallel sequencing was performed on an Ion Torrent PGM (Thermo Fisher Scientific). In our analysis we used the Ion Torrent Suite Software v.4 to process our data and we configured the settings to reduce the total number of false positive indels caused by the high frequency of homopolymer sequencing errors . In addition, we visually evaluated the read depth and the percentage of mutated reads for each variant using the Integrative Genomic Viewer (IGV). Finally, validation by MLPA was made to confirm the identified indels. Data were analyzed using the in-house VarAft software version 2.5, which is available online.Targeted gene sequencing (TGS) has been conducted for  patients. Custom design of the genes’ panel was performed using SureDesign (Agilent Technologies Inc.); probes were generated to cover the exons and 15 bp of the surrounding intronic sequences of a total of 87 candidate genes known to be involved in DNA repair disorders. Library preparation for NGS was performed using Agilent’s HaloPlexIn this study, we report on the clinical, cytogenetic, and molecular investigation of 22 families including 29 FA patients. Our cohort is composed of 8 girls and 21 boys (sex ratio = 2.625) aged from 1 to 28 years. Inbreeding examination showed that 25 patients (86.20%) were born from consanguineous unions. Eight families are multiplex (having more than one affected child). Geographical distribution showed that five patients are from Libya, one from Algeria, one Algerian/Tunisian patient  and 22 are Tunisians. The latest group (Tunisian patients) is composed of 14 patients originating from Southern Tunisia, six from Central Tunisia and two from Northern Tunisia.Most of patients presented typical clinical features of FA including: skin pigmentation abnormalities (86%), microcephaly (52%), triangular face (31%), and skeletal malformations (34%). Other clinical abnormalities, such as ear abnormalities and hearing problems were presented in two patients (F032/16 and F076/15).Chromosomal breakage analysis was successful in all patients except for two patients (GF78/09 and SFA2), for whom blood cell culture showed no growth. For these two patients, results of the molecular analysis confirmed the FA diagnosis. For the others, cytogenetic results revealed a high frequency of chromosomal breakages  in 27 patients when compared to controls .The molecular approach allowed us to identify seven different pathogenic variants at homozygous and heterozygous states. These variants include large deletions of one or two exons  respectively, one nonsense mutation  in exon 28, a small deletion  in exon 38, one likely pathogenic missense variant  in exon 14 that was already associated to FA , a previThe last intronic mutation was inherited at a homozygous state in all FA patients originating from Kairouan . Furthermore, all these patients shared the same haplotype  and another Libyan patient (F075/03) who were both homozygous for the novel missense variant c.1258G > A; p.Asp409Glu.Haplotype findings showed that the patient (SFA15) shares the same homozygous haplotype described to be associated with the founder deletion of exon 15 in Tunisian patients . On the Deletion of exons 4 and 5 has been identified in five patients originating from Sfax. Four of them belong to two related families (family 10 and family 11) and were homozygous for this large deletion. The other patient (GF06/09) was heterozygous and the second mutation is still unknown.Our findings showed that deletion of exon 15, the large deletion of exons 4 and 5, and the intronic mutation c.2222 + 166G > A are founder mutations in Sidi Bouzid/Gafsa, Sfax, and Kairouan, respectively .A summary of genealogical, clinical, cytogenetic, and molecular characteristics of the 29 studied patients is presented in FANCA gene. Indeed, a correlation study between the phenotype of FA patients and the different complementation groups revealed a significantly higher frequency of facial dysmorphia in patients from groups A, G, and C  associated with FA in Tunisia : one reported missense mutation , a novel missense variant , a nonsense mutation , a small deletion (c.3788-3790delTCT), and an intronic mutation (c.2222 + 166G>A). Some identified variants were described in other populations  are due to founder mutations . This hiFANCA variants  are shared among Tunisian, Libyan, and Algerian patients. These findings enhance the previous observations that NA patients share a common ethnic background and consequently the same mutational spectrum . However, the second mutated allele failed to be identified for the rest of patients (four patients: 13.79%). Taking together, we can conclude that the combination between MLPA Sanger sequencing and, possibly, TGS methods for ts in NA . For patThe assignment of NA patients to their genetic FA subtypes and the identification of their respective mutations provide valuable data for a better knowledge of the genetic basis of FA in NA and for a more appropriate management of patients at both preventive  and therapeutic (selection of suitable donors for hematopoietic stem cell transplantation) levels.Processed data related to Sanger sequencing, haplotype analysis and MLPA are available in the article. Raw data related to TGS are available from the corresponding author upon reasonable request. Indeed, in Tunisia, genetic data are considered as personal private data, for these reasons we have submitted the minimal dataset as supporting files but we are not allowed to submit the full raw data. The full raw data may be made available upon request by other investigators and after approval of our IRB.This work was supported by the Ethical Committee of the Institut Pasteur de Tunis (IPT) N° 2017/14/I/LR11IPT05/V0. Written informed consent for publication of both clinical data and photographs was obtained from our patients’ families. Informed consent of the legal representatives of the patient was obtained.AA, FM, MO, and SH established the clinical diagnosis. AB and FT collected the samples. WA and AA analyzed cytogenetic results. AB and FT extracted DNA and carried out the experimental work. OM, SE, AS-G, VD, and NL analyzed molecular data. AB drafted the manuscript. AB, SA, AA, OM, MB, and JS designed the project and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, it is unknown whether there is a change in dysbiosis across the stages of diabetic chronic kidney disease. We investigated a cross-sectional study of patients with early and late diabetes associated chronic kidney disease to identify possible microbial differences between these two groups and across each of the stages of diabetic chronic kidney disease. (2) Methods: This cross-sectional study recruited 95 adults. DNA extracted from collected stool samples were used for 16S rRNA sequencing to identify the bacterial community in the gut. (3) Results: The phylum Firmicutes was the most abundant and its mean relative abundance was similar in the early and late chronic kidney disease group, 45.99 ± 0.58% and 49.39 ± 0.55%, respectively. The mean relative abundance for family Bacteroidaceae, was also similar in the early and late group, 29.15 ± 2.02% and 29.16 ± 1.70%, respectively. The lower abundance of Prevotellaceae remained similar across both the early 3.87 ± 1.66% and late 3.36 ± 0.98% diabetic chronic kidney disease groups. (4) Conclusions: The data arising from our cohort of individuals with diabetes associated chronic kidney disease show a predominance of phyla Firmicutes and Bacteroidetes. The families Ruminococcaceae and Bacteroidaceae represent the highest abundance, while the beneficial Prevotellaceae family were reduced in abundance. The most interesting observation is that the relative abundance of these gut microbes does not change across the early and late stages of diabetic chronic kidney disease, suggesting that this is an early event in the development of diabetes associated chronic kidney disease. We hypothesise that the dysbiotic microbiome acquired during the early stages of diabetic chronic kidney disease remains relatively stable and is only one of many risk factors that influence progressive kidney dysfunction. The human gut harbors a complex community in excess of 100 trillion microbial cells that constitute the gut microbiota. They form a dynamic and symbiotic ecosystem that is in constant interaction with the host metabolism influencing nutrition, physiology, and immune function . The micDiabetes and associated micro and macrovascular complications have reached pandemic proportions, with a global prevalence of 463 million . More thSeveral studies have reported compositional change in intestinal microbiota in humans with diabetes compared to control subjects , and exaClostridium spp. and Bacteroides spp. [Clostridium species in CKD [Research in experimental animal models of chronic kidney disease (CKD) ,15 and ides spp. . It is ks in CKD . This ‘uA further hallmark of intestinal dysbiosis is the reduction of beneficial saccharolytic microbial families, such as Prevetoellaceae, that produce short-chain fatty acids such as butyrate. These short-chain fatty acids help maintain the integrity of the intestinal barrier, and are reduced in individuals with CKD compared to healthy subjects . IndividDespite the disease burden, the pathophysiologic mechanisms underpinning the progression of diabetic CKD are poorly understood. Diabetic CKD is characterised by hypertension, albuminuria, and progressive decline in kidney function, measured as a change in the estimated glomerular filtration rate (eGFR). The role of the gut microbiome in the progression of diabetic CKD has emerged as an area of scientific interest . Individ2), 2 (eGFR 60–89 mL/min/1.73 m2), 3a (eGFR 45–59 mL/min/1.73 m2), 3b (eGFR 30–44 mL/min/1.73 m2), 4 (eGFR 20–29 mL/min/1.73 m2), and 5 (eGFR < 20 mL/min/1.73 m2). The risk of death from any cause, cardiovascular events, or hospitalization increases exponentially from Stage 3b onwards [This prospective, cross-sectional study recruited 95 adults, over a 6 month period, at the time of their outpatient endocrinology clinic visit. The patients consented to providing a stool sample for DNA isolation, in addition to their routine blood tests at the outpatient visit. These patients had diabetes with varying stages (stages 1–5) of chronic kidney disease. Patients were divided into 2 distinct groups: ‘early CKD’ and ‘late CKD’. The early diabetic CKD group was defined as participants with diabetes who had stage 1, 2, or 3a CKD, while the late diabetic CKD group was defined as participants with diabetes who had stage 3b, 4, or 5 CKD. Diabetes-associated chronic kidney disease is the preferred terminology encompassing the range of histological kidney lesions that may be observed . CKD is  onwards .Data collection occurred only at one time point and included information on blood pressure, medical comorbidities, duration of diabetes, the stage of CKD, and its associated complications, medications, and pathology results. Anthropometric data and stool samples were collected on the day of the clinic visit while the remainder of the patient’s information was gathered via access to the Austin Health’s electronic medical record. All of this selected information was then entered into a database specific for this research study for subsequent analysis. There were 70 participants in the early diabetic CKD group and 25 in the late group. Participants provided written informed consent and the study was approved by the Human Research Ethics Committee of Austin Health, Victoria, Australia  and the Human Research Ethics Committee of Deakin University, Australia. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2013.Following collection by the donor in a 50 mL specimen container, samples were aliquoted approximately 0.5–1 g into smaller 1.5 mL Eppendorf tubes before freezing at −80 °C for future DNA extraction.® DNA Stool Mini Kit  according to the manufacturer’s protocol. DNA quantification and purification was assessed using a Qubit fluorometer (Invitrogen). DNA extractions from the stool aliquots were performed at the completion of patient recruitment.DNA was extracted using the Qiagen QIAampPCR amplification and sequencing was performed by the Australian Genome Research Facility . PCR amplicons were generated using the primers and conditions outlined in Paired-ends reads were assembled by aligning the forward and reverse reads using PEAR (version 0.9.5) . PrimersThe categorical variables for the patient characteristics were tested using Fisher’s Exact and the continuous variables were tested with Wilcoxon-signed rank test.Data were pruned to remove representatives classified to Archaea (N = 4), Chloroplast (N = 42), and 22 unassigned OTUs implemented in the package ‘phyloseq’ in the R statistical program version 1.26.1 . We alsoboxplot command in base R. Because our data have potential outliers and were not normally distributed, differences between sample group means were tested with the non-parametric Kruskal-Wallis test, using the command kruskal.test [We calculated the number of observed OTUs and compared the alpha diversity (within the sample) of individuals grouped by the CKD stage using the Shannon Index , which akal.test .adonis command from the package ‘vegan’ [p-values <0.05.For beta diversity (between samples), we used a log transformation of data scaled to read depth per sample and then calculated and plotted weighted UniFrac , which p ‘vegan’  to perfo ‘vegan’ . ResultsClinical and Biochemical Characteristics2 in the early diabetic chronic kidney disease group , and 24.48 mL/min/1.73m2 in the late diabetic chronic kidney disease individuals . This reached statistical significance with a p value < 0.001 (Wilcoxon Signed Rank Test). The mean age in the early CKD group was significantly younger at 66.24 years compared with 72.68 years in the late CKD group . The proportion of the 95 recruited patients in each stage of diabetic CKD is illustrated in After sample quality control, data were available for 95 patient samples. The clinical and biochemical characteristics of the 95 study population are shown in p value = 0.70, The beta diversity indices are used to describe ecological diversity between microbial community samples. The beta diversity of the gut microbiome did not significantly differ between early versus late groups of diabetic CKD individuals or between each of the stages of diabetic CKD when compared with another  or between each of the stages of diabetic CKD when compared with another  of bacterial operational taxonomic units (OTUs) identified to the taxonomic level of phylum in the gut microbiome of individuals with different stages of CKD disease were represented by the phyla Bacteroidetes and Firmicutes. The phylum Firmicutes was the most abundant and its mean relative abundance was similar in the early  and late  CKD group, accounting for 45.99 ± 0.58% in early CKD and 49.39 ± 0.55% in late CKD. Likewise, the mean relative abundance for phylum Bacteroidetes was similar in early diabetic CKD accounting for 42.86 ± 1.40% and 41.20 ± 1.12% in the late diabetic CKD group A. At theFaecalibacterium, Bifidobacterium, Bacteroides, and Akkermansia, which are known to be negatively associated with type 2 diabetes [Prevotella in the family Prevotellaceae showed no significant difference in relative abundance in either early or late diabetic CKD  to a more pathogenic proteolytic fermentation pattern. Although the mechanisms for dysbiosis in diabetes are less clearly understood, it has been shown that individuals with diabetes generally exhibit reduced bacterial diversity characterised by a reduction of butyrate-producing bacteria such as Faecalibacterium prausnitzii and Roseburia intestinalis together with an increase in opportunistic pathogens [Diabetic CKD is a multi-systemic disease process with complex pathophysiological processes. Indeed the cause-consequence relationship between such a dynamic pathological process and associated changes to intestinal microbiota is difficult to differentiate. Factors known to be responsible for the dysbiosis in CKD include increased intestinal barrier permeability secondary to an inflammatory and uraemic milieu associated with CKD and subsequent translocation of pathogenic bacteria and bacterial endotoxins from the gut lumen into the bloodstream due to this increased gut permeability . A majorathogens .The ability to isolate gut dysbiotic causality due to diabetes associated CKD alone, remains a clinical challenge due to the presence of multiple comorbidities such as hypertension, obesity and vascular disease. Furthermore, therapeutic strategies in diabetic CKD include medications and dietary restrictions which as described above, can independently affect the gut microbiome ,45. DespIn particular, the gut microbiome differences between healthy individuals and those individuals with diabetic CKD have been documented ,23,46. WFaecalibacterium, Bifidobacterium, Bacteroides, and Akkermansia [Ruminococcus in individuals with type 2 diabetes [2, and a low GFR subgroup, defined as GFR ≤ 7 mL/min/1.73 m2 [The data has shown that the gut microbiome of individuals with early and late diabetic CKD and across all stages 1–5 of diabetic CKD are similar. Consistent with the literature , we haveermansia  are presdiabetes , also shdiabetes , were prdiabetes  examined/1.73 m2 . Whilst Our data has shown that the average relative abundance of bacterial phyla and family across stages 1–5 of diabetic CKD were mostly similar A,B. ThisPrevotella strains are classically considered to be commensal bacteria due to their extensive presence in the healthy human body. Prevotella is a genus with high genetic diversity within and between species, which could explain its abundance in human healthy microbiota [A distinct gut microbiome with reduced Prevotellaceae families has been associated with CKD . Consistcrobiota . As desccrobiota , maintaicrobiota , and redcrobiota . Similarcrobiota .In our study, there was no demonstrable difference in the operational taxonomic units (OTUs), at any of the taxonomic levels across all stages of diabetic CKD suggesting that dysbiosis appears early in diabetic CKD and persists through to the late stages of the disease thus raising the question of what role gut dysbiosis plays in the progression of diabetic CKD. We postulate that dysbiosis is one of a number of factors that may influence progression of diabetic CKD. Comorbid conditions such as hypertension, diabetes, and vascular disease, together with proteinuria and genetics are some of the contributors to the pathogenesis of chronic kidney disease . More reIt is known that in gut dysbiosis, pathogenic bacteria overgrow and secrete increased amounts of lipopolysaccharides, peptidoglycans, and bacterial DNA into the host circulatory system which are detrimental to intestinal permeability . ConsequDiabetes occurs very commonly and is globally present in pandemic proportions  while diOne of the limitations of this study is its small sample size, especially the presence of only 25 patients with the late stages (3b–5) diabetic CKD resulting in a potential loss of the ability to recognise microbial trends. One of the reasons for this is that the study subjects were selected from an outpatient endocrine clinic, where there is a lesser propensity to see more advanced renal dysfunction. We acknowledge that the inherent problem of a small sample size in the late CKD group together with our large number of covariates, would result in a limited statistical power to detect microbiome differences as well as imprecision of the effect estimate and an elevated false positive rate. Other limitations include the cross-sectional nature of this study cohort, which meant that patient samples were collected at only one time point and not studied longitudinally. Furthermore, our data set did not contain information on antibiotic usage, which is known to have an impact on gut microbiome composition. Another significant limitation is that the dietary habits of the participants were not recorded which will invariably affect the outcome of results. There was also no healthy group, however, as described previously, the microbiome between healthy and diseased group has been well characterised and was not the purpose of this study.Future directions in this area of research should include a longitudinal study of a larger number of patients in each of the stages of diabetic CKD (1–5), with a focus on the predominant form of Type 2 diabetes with information inclusive of dietary intake and medications, especially pertaining to antibiotic use.This study raises some unexpected yet interesting questions about the microbiome in individuals with diabetes and varying kidney dysfunction. We know from current literature that there is a significant difference in the gut microbiome between healthy people and those individuals with diabetic CKD. However, within the limitations of this study, we did not observe a significant change in the microbiome across any of the six stages of diabetic CKD. We propose that once patients achieve a state of dysbiosis in early diabetic CKD, this dysbiosis remains relatively the same and could be considered a susceptibility risk factor, which together with other risk factors , influence the progression to late stage diabetic CKD.Bifidobacterium, Bacteroides, Faecalibacterium, and Akkermansia in relation to type 2 diabetes showed a similar relative abundance between early and late CKD groups, as did the positively associated Ruminococcus. We hypothesise that the dysbiotic microbiome acquired during the early stages of diabetic CKD remains relatively static and is only one of many risk factors that influence progressive kidney dysfunction. These findings warrant a further examination in larger patient cohorts followed longitudinally over time with multiple sampling and clinical data inclusive of information on dietary intake and medications. Only then will it be possible to robustly test whether the dysbiotic microbiome composition remains the same or significantly changes with the progression of diabetic chronic kidney disease.In conclusion, the data arising from our cohort of individuals with diabetes associated with CKD show a predominance of phyla Firmicutes and Bacteroidetes. The families Ruminococcaceae and Bacteroidaceae represent the highest abundance, while the beneficial Prevotellaceae family was reduced in abundance. The most interesting observation is that the relative abundance of these gut microbes does not change across the stages (1–5) of diabetic CKD, suggesting that this is an early event in the development of diabetes associated CKD. The negatively associated genera


On the basis of FMDV outbreak cases, a cross-sectional study was undertaken to collect samples from January 2019 to March 2020 intended for isolation, serotyping, and molecular detection of FMDV in the study district. The purposive sampling method was applied to select the study area for the reason of the presence of FMD outbreak case report during the study period. Totally, 27 FMD clinical samples were collected from affected study population during field outbreak. Out of 27 samples, 18 of them were inoculated on cultured Baby hamster kidney (BHK-21) monolayer cells, and all 27 samples were tested using conventional RT-PCR and sets of specific universal primers. Finally, the PCR products were visualized with UV illumination and imaged with gel documentation system. n = 13) of these cultures exhibited FMDV-induced cytopathic effect (CPE) and the identified serotype was SAT-2 FMD virus. Out of 27 clinical samples tested by conventional RT-PCR, only 12 FMDV samples were found to be FMDV positive by universal primers. Out of 27 samples detected by conventional RT-PCR, only 12 FMDV samples were found to be FMDV positive by universal primers.  The current study results revealed that out of 18 clinical samples subjected to virus isolation, 72.2% ( Our study finding indicated that FMDV is prevalent in the study area and FMDV serotype SAT-2 was the causality for the outbreaks of the disease in the study area. Hence, region-wise FMD outbreak investigation, further phylogenetic analysis, and vaccine matching field isolates should be carried out for effective vaccine development to control the disease. Ethiopia owns the largest livestock population in Africa with an estimated animals' number of 59.5 million heads of cattle, 30.5 million sheep, 30.3 million goats, and 54.5 million chickens . The rolAphthovirus within the family Picornaviridae. Clinically, FMD is manifested by fever, loss of appetite, salivation, vesicular eruptions in mucosa of the mouth, skin of the interdigital spaces and coronary bands of the feet and teats, and sudden death of young stock [Livestock diseases remain the most vital impediments to the development of the sector through reducing production and productivity that could ultimately affect regional, national, and international trade in live animal and animal products' . According stock , 11. As ng stock . Pastorang stock , 13. Genng stock , 15.Foot and mouth disease virus (FMDV) has seven immunologically and genetically distinct serotypes -1, SAT-2, and SAT-3) that cause indistinguishable clinical disease . Within Despite occurrence of several outbreaks of FMDV in the Afar region, there is no even single documented information to know about the disease current serostatus, serotypes circulating in the region in general, and the study area in particular. To develop effective control measures of FMD, determining its serostatus, virus isolation, and identification of the serotype(s) circulating in a particular area would have paramount importance. Moreover, having a detailed knowledge on the specific serotypes circulating in a particular area has paramount importance for companies to target for each specific FMDV serotype for effective vaccine development in steady of rely on production of trivalent vaccine for serotype O, A, and SAT-2.Therefore, the present study was intended for isolation and serotype identification of FMDV from outbreak cases in Aba'ala District of Afar Region from January 2019 to March 2020 in Ethiopia.This research work was implemented in Aba'ala District (Erkudi and Hidmo kebeles), which is located in Afar Region, Ethiopia. This study district was purposively selected for the reason that the presence of active FMD outbreak case report in the course of the study period, January 2019 to March 2020. Afar Regional State shares joint intercontinental borders with Eritrea in the northeast and Djibouti in the east part of the region. The region is described specifically through arid and semiarid weather conditions with low and unpredictable rainfall. The altitude of the region ranges from 120 m below sea level in Danakil depression to 1500 m above sea level. Majority of the pastoral community mainly depend on livestock production for their livelihood. According to APADB (2006), approximately there are 1.9 million cattle population in the region, and 90% of the study animals are managed under pastoral production and the rest 10% in the agropastoral production system. The study area is situated in the north area of the region, northeastern Ethiopia. It lies around between 13°15′ and 13°30′ 1atitude and 39°39′ and 39°55′ longitude. The high temperature of Afar ranges as of 25°C in case of the rainy period to 48°C during the dry season .The study populations were cattle that had experienced outbreak cases of FMDV and manifested typical FMD clinical signs in the Abaʼala district area during the study period of this research work. The study animals were cautiously inspected for the manifestation of distinguishing clinical signs of FMD such as vesicular lesions around the oral cavity, on the feet, salivation, lameness, anorexia, and rise in temperature . All agePrior to field-level investigation and sample collection, district- and kebele-level animal health expertises were informed to report for regional veterinary laboratory centers when FMD outbreak occurred. Therefore, based on the occurrence of active FMD outbreak case report and active outbreak finding, a cross-sectional study was used to collect tissue samples. Clinically, FMD-suspected study populations were physically inspected for the manifestation of FMD with typical signs were sampled to collect biopsy samples that were intended for viral isolation, molecular detection, and serotype identification purpose.The purposive sampling method was employed to select FMD-affected study district, cattle herds, and sampling animals as a result of the occurrence of FMD active case reports in the course of the study period, January 2019 to March 2020. Accordingly, within the study areas (subunits), animals with clear signs and symptoms and animals which are suspected to be infected with FMDV as indicated in A total of 27 representative samples  were collected with the help of tissue forceps from unruptured and freshly ruptured vesicles of clinically affected animals during the course of field outbreak to isolate the circulating viruses responsible for the occurrence of disease. These collected FMD-suspected samples were kept in a sampling bottle containing a virus transport medium that has equal volume of 0.04 M phosphate buffer saline (PBS) with 50% glycerol enriched by antibiotics and antifungal according to the protocol recommended by OIE . Collect2 tissue culture flasks and incubated at 37°C for 1 hr for adsorption of the virus. Then, cell cultures were added 8 ml of the maintenance medium (2% MEM) and incubated at 37°C and 5% CO2 in a humidified incubator. The appearance of virus-induced cytopathic effect (CPE) was observed daily under the inverted microscope. The inoculated cell line was harvested when 85–100% of CPE was observed. These infected cells did not show CPE within 72-hour postinfection on the third passage were supposed to be virus negative [The samples collected were processed and cultured on bovine hamster kidney (BHK-21) cell monolayer with three subsequent passages as follows. About 1 gram of each tissue was taken and washed three times using sterile phosphate buffered saline containing antibiotics and antifungal (PBS) on Petridish. Then, washed tissues were transferred to sterile mortar, cut into pieces using scissor and minced by scalpel blade. These minced tissues were then grounded and homogenized in sterile sand with a sterile pestle and mortal. Nine ml of PBS was added to the homogenized tissues and well mixed, and small volume tissue culture made and small amount of five percent antibiotics  containing the medium were added so that the final volume was ten times that of the epithelial tissue, producing of ten percent suspension . All pronegative , 24. Samnegative .FMD serotyping was executed using both antigen detection sandwich ELISA and sets of serotype specific primers intended for testing of FMD virus and identifying the serotypes responsible for outbreaks cases. Sandwich‐ELISA was executed with particular combinations of anti‐FMDV monoclonal antibodies (MAb), used as coated and conjugated antibodies. The kit was developed for detection and serotyping of FMDV O, A, C, SAT-1, and SAT-2. A pan‐FMDV test, detecting any isolates of O, A, C, Asia1, and SAT serotypes, was also included in the kit to complement the specific serotyping of FMDV. The test was implemented based on the manufacturer's instruction and OIE . A totalμl of dilute buffer was dispensed into all wells of the test plate, and then 25 μl of previously diluted samples using ELISA buffer and ready‐to‐use controls was dispensed into the appropriate wells of the test plate precoated with recombinant FMD viral antibody. One positive control for each FMD types O, A, SAT-1, and SAT-2 and negative controls were included in each plate. The plates were sealed using the enclosed plate sealer and incubated for 1 hr at room temperature (20–25°C). After incubation, all fluids on the plates were discarded and the remaining residual fluids were removed. Then, 200 μl of washing solution was added and incubated for 3 min at room temperature; subsequently, wells were emptied and the washing was repeated twice . Then, all residual fluids were removed by tapping on clean absorbent paper and 50 μl of conjugate. A was added from columns 1 to 8, and the same volume of conjugate B was added from columns 9 to 12. Plates were covered and incubated at room temperature for 1 hour. After incubation, 50 μl of substrate per well was added to all wells and plates were covered and left at room temperature for 20 minutes in the dark. The reaction was stopped by adding 50 μl of stop solution (sulfuric acid (H2SO4)). Immediately after stopping, reading the optical density (OD) of each well was done at 450 nm wavelength using microplate reader.About 25 The presence of FMD viral genetic material in all 27 collected field samples was tested using conventional RT-PCR and specific primers that amplify viral protein 1 (VP1) of FMDV using the RNeasy Mini Kit following the manufacturer's instruction .μl buffer AVL carrier RNA in the mircocentrifuge. The tubes were briefly centrifuged to remove drops from the inside of the lid. Then, 560 μl of ethanol (70%) was added to the sample and mixed by pulse vortexing for 15 seconds followed by centrifuging to remove drops from the inside lid. Then, 630 μl of the solution was applied to the QIAMP Mini-spin column in a 2 ml collection tube and centrifuged 12,500 rpm for 1 min. The filtrate was discarded, and the column was placed in a fresh 2 ml collection tube. Then, 500 μl of buffer AW2 were added and centrifuged at 12,500 rpm for three min and the filtrate was discarded. Next, 65 μl of Buffer AVE was added to the column equilibrated at room temperature for one min and centrifuged at 12,500 for 1 min. Using reverse transcription polymerase chain reaction (RT-PCR) and specific primers set FMDV7-forward (FMDV7F) and FMDV7-reverse (FMDV7R) as depicted in Total RNA was extracted from collected FMD-suspected clinical sample suspension using Qiagen RNA extraction kit following the manufacturer's instructions as . Brieflyμl gel red with loading dye, then the PCR product was loaded in the volume of 10 μl in each well, and 10 μl molecular marker (ladder) was added started 100 bp plus. Electrophoresis was run for one hour at 120 V. Then, the DNA band was visualized by UV illumination, using desktop according to the base pair (bp), and then the size was determined and documented.The PCR products were analyzed on the prepared 1.5% Agarose gel by adding 4 Data generated from laboratory investigations were recorded and coded using Microsoft Excel spreadsheet and analyzed using STATA version 14.2 for Windows. Cell-culture results, CPE development, and molecular characterization results were recorded and tabulated. Virus isolation and molecular detection of FMD virus were elaborated using descriptive statistics analysis. Moreover, regarding the molecular characterization, the banding patterns of individual sample were scored based on the presence or absence of the bands with the appropriate base pairs.In this outbreak investigation and sample collection, characteristic clinical signs of FMDV in the study population were salivation, lameness, vesicle formations on oral cavity, and interdigital vesicles. Suggestive lesions of FMD on the mouth contained destructions and sores on the upper and lower pad area and tongue, while feet abrasions consist of wearing away on the interdigital space. These study populations were reluctant to travel and lagging behind the healthy cattle and deny for feeding.n = 13) representative samples exhibited morphological alterations (FMDV cytopathic effect (CPE) on BHK-21 cells, while the other clinical samples of FMDV (n = 9) did not inoculated on the cells because these samples were collected from the same outbreak in the study areas. Out of 13 FMD clinical samples that showed cytopathic effect on BHK-21 cells, 33.3% (n = 6), 22.2% (n = 4), and 16.7% (n = 3) were epithelial tissues, vesicular fluid, and swab samples, respectively. These FMD positive clinical samples were characterized primarily by a quick sloughing of BHK-21 monolayer cells and these sloughed cells were roughly round, swelling, and formed singly in shape  is responsible for frequent outbreaks and causes significant economic devastation in the region in particular and on the nation in general. The disease is described by development of typical FMD lesions around the mouth as well as on the foot and unexpected losses of newborn calves , 11. Occn = 13) field samples showed FMDV-induced cytopathic effect (CPE). These cells were appeared as rounding in cell culture, swelling, and clumping of the cells as one can demonstrate from In this research finding, from 18 suspected clinical samples subjected to BHK-21 cell line adaptation, 72.2%  were found to be positive. Of these 12 samples detected as positive for FMDV, bovine epithelial tissues were accounted for 22.2% (n = 6) and had the lower Ct values which could indicate higher concentrations of the virus in these samples. Our results also showed that bovine vesicular fluid samples were accounted for 14.5% (n = 4) and swab samples were accounted for 7.4% (n = 2). This study finding confirmed the existence of more FMD viral RNA in the epithelial tissue samples as compared to vesicular and swab samples. This study finding is supported by OIE [In this study, out of 27 clinical samples detected using conventional PCR for the presence of FMDV genetic material in the sample, only 44.4% (d by OIE  as this d by OIE .The present study finding indicated that FMDV is prevalent in the study area of Afar Region as confirmed by clinically, serologically, and molecular techniques particularly in the study area of the region and serotype SAT-2 was the causality for the outbreaks of the disease in the study area. The occurrence of this disease is a foremost badly behaved for the improvement of the livestock industry as it causes enormous worldwide harms of livestock sector as well as severe impacts on export earing from national and international trade thereby threaten the living means of livestock keepers in particular and income source of the country in general. Out of the serotypes identified in our country, the identified prevailing serotype was SAT-2 that causes frequent outbreaks in the study area of Afar Region, Ethiopia. Region-wise regular FMD outbreak investigation to have more full information about the serotypes, topotypes involving in the region, and vaccine matching studies of field isolates to evaluate vaccine protection potential has paramount important for effective vaccine development.


In the successful transition towards a circular materials economy, the implementation of biobased and biodegradable plastics is a major prerequisite. To prevent the accumulation of plastic material in the open environment, plastic products should be both recyclable and biodegradable. Research and development actions in the past few decades have led to the commercial availability of a number of polymers that fulfil both end-of-life routes. However, these biobased and biodegradable polymers typically have mechanical properties that are not on par with the non-biodegradable plastic products they intend to replace. This can be improved using particulate mineral fillers such as talc, calcium carbonate, kaolin, and mica. This study shows that composites thereof with polybutylene succinate (PBS), polyhydroxybutyrate-hexanoate (PHBH), polybutylene succinate adipate (PBSA), and polybutylene adipate terephthalate (PBAT) as matrix polymers result in plastic materials with mechanical properties ranging from tough elastic towards strong and rigid. It is demonstrated that the balance between the Young’s modulus and the impact resistance for this set of polymer composites is subtle, but a select number of investigated compositions yield a combination of industrially relevant mechanical characteristics. Finally, it is shown that the inclusion of mineral fillers into biodegradable polymers does not negate the microbial disintegration of these polymers, although the nature of the filler does affect the biodegradation rate of the matrix polymer. The development of plastic materials in the 20th century has led to a multitude of plastic products that combine lightweight, high performing mechanical properties and outstanding barrier properties with the ability to be molded in virtually any shape imaginable. As a result, plastic products have become an indispensable part of human life as they are found in applications such as food packaging, clothing, home appliances, and toys. The downside of this innovation is that society now faces a huge challenge to properly dispose and recycle these plastic products and prevent the accumulation of plastics in the natural environment. Even though the need for a functional recycling infrastructure is clear, which is reflected in the development of new and improved waste collection, sorting, and recycling  technologies, there is still an unacceptable large fraction of plastic waste that ends up in the natural environment . As a rePolybutylene succinate (PBS) BioPBS FZ91PB and polybutylene succinate adipate (PBSA) BioPBS FD92PB were obtained from MCPP, Dusseldorf, Germany. Polybutylene adipate terephthalate (PBAT) Technipol 061/E was obtained from Sipol, Mortara, Italy. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) Aonilex X331N was obtained from Kaneka, Westerlo-Oevel, Belgium. Talc Luzenac A10HC powder (d50% = 2.1 μm) and Mica 200k powder (d50% = 60 μm) were obtained from Imerys, Toulouse, France. Calcium carbonated chalk VS10 powder (d50% = 1.8 μm) was obtained from Omya, Oftringen, Switzerland. Kaolin Burgess Iceberg powder (d50% = 1.4 μm) was obtained from Burgess Pigment, Sanderville, GA, US.All polymers were dried at 83 °C for at least 8 h in a Gerco two-chamber dry-air desiccant dryer  to achieve moisture contents below 250 ppm prior to further processing. Extrusion was performed with a Berstorff ZE25x40D twin screw co-rotating extruder  at maximum temperatures of 180, 180, 160, and 180 °C for PBS, PBSA, PBAT, and PHBH, respectively. Injection molding was performed with a Sumitomo Demag IntElect 2  at maximum temperatures of 170, 170, 160, and 180 °C for PBS, PBSA, PBAT, and PHBH, respectively. Tensile bars (75 mm × 5 mm × 2 mm) were produced conforming to ISO527-2 and impact bars (80 mm × 10 mm × 4 mm) were produced according to ISO 294. Sheet extrusion was performed with a Dr. Collin single screw extruder  with a sheet die at maximum temperatures of 180, 180, 135, and 175 °C for PBS, PBSA, PBAT, and PHBH, respectively.Mechanical properties of the polymer compounds were evaluated using a Zwick Z010 tester with Multisens extenso meters , which was built according to ISO 527. Tests were performed following standards ISO 527-2 (for injection molded samples) and ISO 527-3 (for sheets and films). Measurements were performed in 5-fold. The test speed for determination of the Young’s modulus was 1 and 10 mm/min for determination of the strength at break and strain at break. Charpy impact resistance of the polymer compounds was evaluated using a Ceast 9050 tester  equipped with a Charpy 4J hammer which was built according to ISO 179 and conforming to the method based on the standards ISO 179-1eU (notched).GPC measurements to determine molar mass were performed using a Viscotech VE 2001 GPC max provided with a TDA305 Triple Detector Array . Columns used were a PSS PFG analytical linear M and guard column, molecular range ~250–2.5∙10e6 D (PMMA in HFIP). The selected solvent was hexafluoroisopropanol (HFIP) with 0.02 M potassium trifluoroacetate (KTFA). All measurements were performed in duplicate. The Melt Flow Index (MFI) was determined using a Zwick MFlow tester according to ISO 1133 and conforming to the methods described in ASTM D1238 and ISO 1133.Assessment of the biodegradation in soil was based on the method described in ASTM G160-12(2019) .Dumbbell-shaped samples were cut from sheets and were exposed to soil under laboratory-controlled conditions. The soil, a sandy topsoil obtained from organic experimental and training Farm “Droevendaal” , was inoculated with 2% (based on dry weight) fresh, mature compost from an industrial composting facility treating the organic fraction of municipal solid waste. Containers (40 cm × 16 cm × 18 cm) with buried samples were incubated at constant temperature (25 ± 2 °C) and relative humidity (90 ± 5%). Moisture content of the soil was maintained constant at 80% of the water holding capacity by spraying demineralized water to correct for any evaporation during the incubation. Viability of the soil was regularly checked with an untreated cotton cloth reference, which should lose >50% of the initial tensile strength in 5 days of exposure to the soil. In addition, soil pH was monitored as well.Plastic samples were recovered from the soil at regular intervals; for this purpose, 6 replicate samples were buried per datapoint. Recovered samples were carefully rinsed and cleaned to remove the soil and subsequently photographed. Prior to mechanical analyses, the samples were conditioned for 1 week at 50% RH and 23 °C. Control samples were recovered after of 1 day of exposure to exclude any influence of the burial and cleaning procedure.PBS was selected as one of the model matrices to compare the effect of different types of inorganic mineral fillers on the mechanical properties of biodegradable polymer composites. The non-reinforced polymer has properties in the same order of magnitude as PP, although the modulus is typically a factor 2–3 too low for adequate material substitution in current non-biodegradable applications. Furthermore, several applications benefit from an outstanding water vapor barrier capacity and optical properties, in which PP clearly outperforms PBS, but these properties are not included in this overview. The effect of talc, calcium carbonate, kaolin, and mica fillers on the selected mechanical properties  of PBS is depicted in In polymer composite engineering , an increase in modulus is typically accompanied with a reduced impact resistance. In order to facilitate material selection, it is insightful to compare these properties to show the relation between these counteracting material properties comparable to the well-known Ashby diagrams . Figure 2) in applications that mainly rely on mechanical performance, this figure indicates that PBS filled with 10–30 wt.% talc might be a suitable biodegradable alternative. Only when considering applications in which very high moduli are required, it might be favorable to select a different filler such as mica. At talc and mica filler concentrations around 50 wt.%, moduli and impact resistances in the range of PLA (3000–4000 MPa and 2–4 kJ/m2) are obtained. Based on this analysis, the resulting polymer composites could be considered as a PLA alternative that has a higher biodegradation rate in natural environments such as soil. However, this will depend on the targeted application as it is anticipated that complex processing operations such as thin-walled injection molding and sheet extrusion are severely complicated at these high filler ratios.Based on the comparison in An injection molding PHBH grade was selected as a second model polymer matrix due to the substantially different mechanical performance compared to PBS. In addition, the selected compound is certified marine biodegradable (OK biodegradable MARINE ). Figure2), several of the composites developed in this work could yield a biodegradable alternative.As PBSA and PBAT are less rigid polymers than PHBH and PBS, they possess lower moduli and strength and a higher strain at break and impact resistance in their unfilled form. The polymers investigated in this study all show biodegradation rates in soil that are substantially higher than those of conventional non-biodegradable polyolefins  and polyesters . This material property gives these polymers an additional end-of-life scenario that can come into effect when products made from these materials intentionally or unintentionally end up in the natural environment. However, as it shown in this study that the use of mineral fillers can be used to direct their mechanical properties towards those of industrially applied plastics, it is crucial to identify whether these additives do not negate their unique biodegrading features. The full characterization of polymer composite biodegradability requires monitoring of the actual demineralization process into water and carbon dioxide, which is beyond the scope of this study. Nevertheless, a soil disintegration test was performed to identify the effect of both calcium carbonate and talc on the soil biodegradation of PHBH. The visual disintegration of three PHBH compounds upon soil exposure in a timeframe of 8 weeks is depicted in Aside from the visual observations in This study gives a comprehensive overview of the effect of various particulate mineral fillers  within PBS, PHBH, PBSA, and PBAT polymers. Thereby, plastic materials with mechanical properties ranging from tough elastic towards strong and rigid are obtained. It is indicated that the balance between the Young’s modulus and the impact resistance for this set of polymer composites is subtle. Still, a select number of compounds yield a combination of industrially relevant mechanical characteristics. Finally, it is shown that the inclusion of mineral fillers into biodegradable polymers does not negate the microbial disintegration of these polymers and that the nature of the filler does affect the biodegradation rate of the matrix polymer.


It appeared that, except for the MC4R SNP, all the other polymorphisms were found to be associated with various laboratory parameters. Arsenic concentration in urine was associated with all four CNR1 and LEP SNPs, while cadmium concentration in blood was affected by the VDR polymorphism. Moreover, some significant relationships were also observed between CNR1 rs1049353 and FTO rs9939609 gene variants and markers of lead exposure. These results imply SNPs within genes coding for proteins involved in development of metabolic syndrome may be of prognostic value for persons directly exposed to lead, cadmium and arsenic.The purpose of this work was to assess the influence of selected CNR1, MC4R, LEP, FTO and VDR FOKI gene polymorphisms on blood and urine concentration markers of lead, cadmium and arsenic in a population directly exposed to these metals. Eighty-five people exposed to lead, arsenic and cadmium were qualified to take part in the study. Standard urine samples and 25 mL of venous blood from each worker were collected to assay basic laboratory and toxicological markers as well as selected single nucleotide polymorphisms (SNPs) within CNR1—cannabinoid receptor 1 gene ( Metabolic syndrome is an important global clinical problem and a challenge for modern medicine . MetabolResearch that aims to explain the causes of metabolic syndrome and its epidemic occurrence is based, on the one hand, on genetic and genotypic analyses and, on the other hand, observation of influence of the external environment ,5.Metabolic syndrome is multi-genetic. Among the genes with the strongest relationship with metabolic syndrome, CNR1—cannabinoid receptor 1 gene, LEP—leptin promoter gene, FTO—alpha-ketoglutarate-dependent dioxygenase gene, MC4R—melanocortin 4 receptor gene and VDR—vitamin D receptor stand out.rs806381, rs806368, rs1049353, rs12720071), as well as several possible combinations. The most popular, A to G transition in rs1049353, leads to higher body mass index and wider waist circumference [rs17782313) tends to be associated with higher body mass index and insulin resistance [CNR1 (cannabinoid receptor 1) is a G protein-coupled receptor activated by endogenous and exogenous cannabinoids. They are found mostly in the central nervous system, in the cerebellum, nucleus accumbens and several other brain regions responsible for hunger, satiety and the reward system . Severalsistance . FinallyAccording to current knowledge, exposure to metals may affect the development of metabolic syndrome. There are more and more reports confirming the existence of such a relationship.Using data from the 2011–2014 National Health and Nutrition Examination Survey, Bulka et al. evaluated associations between essential and toxic metals exposure and metabolic syndrome . The posOn the contrary, an analysis based on the Korea National Health and Nutrition Examination Survey (KNHANES) found that a higher prevalence of metabolic syndrome was associated with higher blood lead levels in the Korean population .In a study by Wang et al., blood and urinary markers of 18 heavy metals among 9537 adults in NHANES 2003–2014 were evaluated. This study suggests that cumulative exposure to heavy metals as mixtures is associated with obesity and its related to chronic conditions such as hypertension and diabetes type II .Luzhetskyi and co-authors proved that children with higher serum levels of cadmium and arsenic (1.4–2.0 times vs. the reference group) demonstrated 2.2 times more frequent endocrine diseases, up to 2.7 times more frequent obesity-related diseases, when compared to the reference group. Metabolic disorders in that group were associated with some lipid metabolism changes .Similarly, study of Kawakami et al. demonstrated that exposure to cadmium caused a reduction of adipocyte size and the modulation of adipokine expression. The reduction in adipocyte size by Cd may arise from an imbalance between lipid synthesis and lipolysis. In addition, the expression levels of leptin, adiponectin and resistin decreased in adipocytes. So, exposure to Cd may induce unusually small adipocytes and modulate the expression of adipokines differently from the case of physiologically small adipocytes, and it may accelerate the risk of developing insulin resistance and type 2 diabetes .Numerous data concerning potential arsenic, cadmium and lead effects on development of obesity and metabolic syndrome have inspired us to pose a hypothesis that concentrations of these metals in people occupationally exposed are related to selected gene polymorphisms and determine metabolic disorders.The purpose of this work was to assess the influence of selected single nucleotide polymorphisms (SNPs) coding for proteins involved in development of metabolic syndrome with the previously mentioned selected gene polymorphisms on blood and urine concentration markers of lead, cadmium and arsenic in a population directly exposed to these metals.Eighty-five people, employees of a copper smelter and refinery, were qualified to be in the study. Inclusion criteria for the study were employment in workplaces exposed to arsenic, cadmium and lead ) and work-related exposure to metals for at least 0.25 years. Size and quality of environmental exposure for all subjects included in the study were similar because all subjects resided in the same region. Only people who lived in the region for a long time (for at least 3 years) were included in the study group. The data and biological sample were collected in June 2016. There were 67 men and 18 women, and age ranged from 26 to 67 years. Thirty-three participants were obese, 34 of them were previously diagnosed with hypertension and 37 of them were smokers. Full clinical characteristics of the group are shown in All the participants were asked to fill in a questionnaire about their medical history and lifestyle. Next, basic anthropometric measurements were taken. We also took standard urine samples and 25 mL of venous blood from each worker just after finishing their work shift to assay basic laboratory and toxicology markers as well as selected single nucleotide polymorphisms.3 and parathormone) were determined by standard methods in accordance with the manufacturer’s instructions.Blood count, fasting glucose, HbA1C, lipids  and calcium-phosphate balance markers  was used. We monitored the accuracy of the analytical method by analyzing samples of a reference material, Seronorm Trace Elements Urine , and participation in an intercomparison program for toxicological analyses in biological materials, G-EQUAS. The biological exposure limit proposed by the National Hygiene Institute and Institute of Occupational Health for arsenic in urine is 35 µg/L (35 µg/g creatinine).We also determined concentrations of lead (Pb-B), cadmium (Cd-B), zinc protoporphyrins in blood (ZnPP) and arsenic in urine (As-U). Blood lead and cadmium concentrations were measured by graphite furnace atomic absorption spectrometry Solaar M6 . The calibration curves of lead and cadmium were prepared with blood standards of certified reference material (BCR IRMM). Both methods are routinely monitored by determination of reference material (Recipe) and participation in an intercomparison program for toxicological analyses in biological materials, G-EQUAS. The measurement was calculated as micrograms per liter (µg/L), and the biological exposure limits were 500 µg/L for lead and 5 µg/L for cadmium according to recommendations of the National Hygiene Institute and Institute of Occupational Health. ZnPP was measured using a rapid fluorometric screening method by means of Hematofluorymeter ProtoFluor . Total urine arsenic concentration was measured by Hydride Generation Atomic Absorption Spectrometry (HGAAS) using the VP100 Continuous Flow Vapour System. To determine the calibration curve, the reference material ClinCalrs17782313 (T>C), located upstream of the MC4R gene on chromosome 18q22; rs7799039 (G>A), a common promoter polymorphic site within the LEP gene on chromosome 7q31.3; rs9939609 (G>A), a SNP within intron 1 of the FTO gene on chromosome 16q12.2; rs10735810 (FokI) (C>T), located within exon 2 of the VDR gene on chromosome 12q11; and four polymorphisms of the CNR1 gene located on chromosome 6q15 , and rs12720071 A>G, a substitution in 3′UTR). Genotyping was performed by real-time polymerase chain reaction (PCR) amplifications and a melting curve analysis using a LightSNiP typing assay . Real-time PCR was carried out on a LightCycler 480 Real-Time PCR system  in accordance with the conditions recommended by the manufacturers.In the study we analyzed selected single nucleotide polymorphisms (SNPs) for cannabinoid receptor 1 gene (CNR1), melanocortin 4 receptor gene (MC4R), leptin (LEP), alpha-ketoglutarate-dependent dioxygenase gene (FTO) and vitamin D receptor FokI. In the present study, the following eight SNPs were selected: The research was compliant with Good Clinical Practice guidelines, The Declaration of Helsinki and was based on consent from a local Bioethical Committee .p < 0.05 were regarded as statistically significant.Statistical analyses were calculated using the statistical program STATISTICA 13 . For the quantitative variables, the arithmetic mean (X), the median value (Me), the standard deviation (SD) as well as the minimal (Min) and maximal (Max) values of assayed parameters were calculated in the studied groups. Distribution of variables was tested using the Lilliefors and W-Shapiro–Wilk tests. In the case of independent, quantitative variables having normal distributions, a t test for independent variables and the analysis of variances ANOVA  were used in the further statistical analysis. The U test of Mann–Whitney or a non-parametric equivalent of Kruskal–Wallis ANOVA analysis of variance test were used in the case of variables with non-normal distributions. The significant differences between the arithmetic means were estimated using a post-hoc Newman–Keuls test. Results for the nominal variables were presented in percentages. In order to assess the relations between studied variables, a correlation analysis was performed. In the case of variables having a normal distribution, Pearson’s r was calculated, and for the variables with a distribution other than normal, the Spearman’s r correlation coefficient was used. Results at the level of The mean blood calcium, phosphorus, 25-hydroxyvitamin D and parathormone concentrations in the occupationally exposed group were accordingly 9.69 ± 0.33 mg/dL, 3.43 ± 0.66 mg/dL, 20.81 ± 7.69 µg/L and 46.68 ± 20.02 ng/L. These are presented in Arsenic concentration was 11.74 ± 9.37 µg/L, cadmium was 0.84 ± 0.80 µg/L, lead was 199.23 ± 117.02 µg/L and ZnPP was 47.94 ± 30.64 μg/dL. A total of 3.5% of employees had a urine arsenic concentration higher than the norm of the allowable concentration in biological material (determined as a maximum of 35 μg/L). Totals of 1.2% and 16.5% of workers, respectively, had a blood lead concentration and blood zinc protoporphyrin concentration higher than the norms of the allowable concentration in biological material . Nobody in the study group was characterized by exceeding the norm of the permissible blood cadmium concentration. These are presented in rs12720071 GG homozygosity was the rarest and was detected in one individual only (1.2%). The other rare genotypes were MC4R rs17782313 GG and CNR1 rs1049353 AA homozygosity observed both in five (5.9%) cases, and the CNR1 rs806386 CC genotype was found in seven (8.2%) cases. These distributions closely resemble those described for other European populations.Distributions of alleles and genotypes of selected SNPs in the studied population are shown in rs806381 in the CNR1 gene is related to a statistically significant lower arsenic concentration, compared to heterozygosity AG and homozygosity GG, and the presence of allele G in this locus is associated with a significantly higher arsenic urine concentration. In locus rs12720071 of the CNR1 gene, homozygotes GG have statistically significant higher arsenic concentrations than heterozygotes AG and homozygotes AA, and allele A in this locus can be associated with a statistically significant lower arsenic concentration. The analysis also has shown that allele A in locus rs1049353 of the CNR1 gene can be responsible for lower arsenic concentrations, and allele G in locus rs7799039 of gene LEP is responsible for significantly higher arsenic concentrations in the studied population.The results of comparative analyses of arsenic urine concentrations of subgroups based on genotype criteria and alleles of single nucleotide polymorphisms of genes CNR1, MC4R, LEP, FTO and VDR FokI are presented in rs10735810 of VDR FokI gene can be a factor responsible for the significantly lower cadmium concentration in the studied population.The results of comparative analyses of cadmium blood concentrations of subgroups based on genotype criteria and alleles of single nucleotide polymorphisms of genes CNR1, MC4R, LEP, FTO and VDR FokI are presented in rs1049353 of the CNR1 gene have a significantly higher blood lead concentration compared to heterozygotes AG and homozygotes AA. The presence of allele A in the locus is correlated with a statistically relevant lower lead blood concentration.Similarly, the results of comparative analyses of lead blood concentrations of subgroups based on genotype criteria and alleles of single nucleotide polymorphisms of genes CNR1, MC4R, LEP, FTO and VDR FokI are presented in rs1049353 of the CNR1 gene may result in a statistically lower ZnPP concentration compared to homozygosity AA and GG, and allele A is responsible for lower ZnPP concentrations. Apart from that, we documented that allele A in locus rs9939609 of the FTO gene is responsible for higher ZnPP concentration.The results of comparative analyses of zinc protoporphyrin blood concentrations of subgroups based on genotype criteria and alleles of single nucleotide polymorphisms of genes CNR1, MC4R, LEP, FTO and VDR FokI are presented in p = 0.040), ZnPP concentration and platelets , ZnPP concentration and phosphorus in blood  as well as ZnPP and vitamin D .In the correlation analysis we found statistically significant, linear correlations between cadmium concentration and white blood cell count  and concentration of lead and ZnPP [We were also able to find that allele T in locus and ZnPP . Our stuand ZnPP .rs1049353 polymorphism was associated with specific changes in brain morphology as well as with evolution of positive symptoms in schizophrenia [In studied population, we were able to determine a significant correlation between the CNR1 polymorphism and lead and ZnPP concentrations. Again, this is the first research proving this relation. However, polymorphism of CNR1 is being thoroughly studied. It was found that ophrenia . This poAs for the linear correlation between cadmium concentration and white blood cell count, our research is compliant with many previous studies, including the most recent ones ,27. Corrrs806381 CNR1 gene and rs7799039 LEP gene, and at the same time a carrier of allele A in loci rs1049353 and rs12720071 CNR1 gene, the increase in As-U concentration due to the impact of the first two alleles may be reduced as a result of the impact of the second two alleles.Finally, it should be mentioned that, when analyzing multiple polymorphisms affecting a single variable , it is necessary to consider the possible compensatory effects of polymorphisms. In the current study, this compensation in individuals may be due to the effect of polymorphisms on urinary arsenic levels. If a person is a carrier of allele G in loci rs12720071 CNR1—18.8%; allele C in locus rs17782313 MC4R—37.6%). As for the first allele, we were able to obtain statistically significant correlation. If statistically important correlations are found in a relatively small group, the correlation is strong and it will be even more visible in larger groups. Finally, showing specific correlations between polymorphisms and metal concentrations is just the first step in understanding their complex role in the human organism; yet, it is an important step forward.The authors of this work see two main limitations of this study. First, there is little or no research to compare and confront our data. Our study was conducted with the highest standards; we found statistically significant correlations, but still, further research needs to be conducted to confirm our results. Secondly, our study is based on a relatively small population (85 persons) for genetic study. This results in poor representation of certain alleles Single nucleotide polymorphisms within genes coding for proteins involved in development of metabolic syndrome may be of prognostic value for persons directly exposed to lead, cadmium and arsenic.(2)rs806381, rs1049353 and rs12720071 of gene CNR1 and polymorphism rs7799039 of gen LEP and arsenic concentration in urine were acquired:rs806381 CNR1 and locus rs7799039 LEP can be responsible for higher arsenic concentrations;Allele G in locus rs1049353 and rs12720071 CNR1 can be responsible for lower arsenic concentrations.Allele A in locus In the group occupationally exposed to arsenic, cadmium and lead, certain associations between polymorphisms (3)rs10735810 VDR FokI gene:rs10735810 VDR FokI gene can be responsible for lower cadmium concentration.Allele T in locus Cadmium concentration in blood in people occupationally exposed can be determined by polymorphism of (4)rs1049353 gene CNR1 and rs9939609 gene FTO and markers of lead exposure (lead and zinc protoporphyrin in blood):rs1049353 CNR1 gene can be responsible for lower lead and ZnPP concentrations;Allele A in locus rs9939609 FTO gene can be responsible for higher ZnPP concentration.Allele A in locus In people occupationally exposed to arsenic, cadmium and lead, there are certain interactions between polymorphisms (5)rs17782813 MC4R gene, as the only one in our study, did not affect concentrations of selected markers amongst workers occupationally exposed to lead, cadmium and arsenic.Polymorphism


A comparison of the microbiota composition at the genus and species levels pointed toward isolates specifically adapted to the two extreme environments. However, genera and species recorded as being similarly abundant in the microbiota of indoor and outdoor hens are equally as noteworthy because these represent microbiota members that are highly adapted to chickens, irrespective of their genetics, feed composition, and living environment.In this study, we compared the caecal microbiota composition of egg-laying hens from commercial production that are kept indoors throughout their whole life with microbiota of hens kept outdoors. The microbiota of outdoor hens consisted of lower numbers of bacterial species than the microbiota of indoor hens. At the phylum level, microbiota of outdoor hens was enriched for Bacteroidetes  and Proteobacteria (9.33 ± 4.99% in outdoor and 5.47 ± 2.24% in indoor hens). On the other hand, Firmicutes were more abundant in the microbiota of indoor hens (33.28 ± 5.11% in indoor and 20.66 ± 4.41% in outdoor hens). Horizontally transferrable antibiotic resistance genes  Chickens in commercial production are hatched without any contact with adult birds, and colonization of their intestinal tract is dependent on environmental sources only. The development of their microbiota is therefore slower than in chicks that have any form of contact with adult hen microbiota ,2. The sAttempts with administration of microbiota from healthy adults to newly hatched chicks in intensive production systems date back to the 1970s . The conIn this study, we therefore compared the caecal microbiota composition in chickens from commercial production, kept indoors throughout their whole life and with microbiota of chickens kept outdoors throughout their whole life. Caecal microbiota was selected due to its high complexity and also importance for natural resistance of chickens to the colonization of pathogens like Salmonella. Private owners in village areas of the Czech Republic keep small flocks of 5–10 hens for their own egg consumption, i.e., not for global market distribution . This coTwenty 30–50-week-old hens were obtained from three different egg producing farms using enriched battery cages for egg production. At any time of rearing or egg production, these hens had no access to the outdoor environment and were kept in a strictly controlled indoor environment. An additional 17 hens were obtained from six different backyard poultry flocks with the agreement of the owners. These hens were 2–4 years old and were reared year-round in an outdoor environment with access to a simple henhouse. Both indoor and outdoor hens were brought to the post-mortem room, humanely sacrificed, and their caecal contents were collected and frozen at −70 °C. The time from euthanization to sample freezing never exceeded 30 min and time between sample freezing and DNA purification never exceeded three months.The handling of animals in the study was performed in accordance with current Czech legislation . The specific experiments were approved by the Ethics Committee of the Veterinary Research Institute followed by the Committee for Animal Welfare of the Ministry of Agriculture of the Czech Republic .TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-MID-GT-CCTACGGGNGGCWGCAG-3′ and reverse primer 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-MID-GT GACTACHVGGGTATCTAATCC-3′. The sequences in italics served for index ligation whereas the underlined sequences allowed for amplification over the V3/V4 region of 16S rRNA genes. MIDs represent different sequences of 5, 6, 7, or 9 base pairs in length, which were used to identify individual samples after the whole sequencing run. PCR (Polymerase Chain Reaction) amplification was performed using a KAPA HiFi Hot Start Ready Mix kit , and the resulting PCR products were purified using AMPure beads . In the next step, the PCR product concentration was determined spectrophotometrically, the DNA was diluted to 100 ng/μL, and groups of 14 PCR products with different MID sequences were indexed with the same index from Nextera XT Index Kit following the manufacturer’s instructions . The next set of 14 PCR products with different MID sequences were indexed with the next index from the Nextera XT Index kit, thus allowing an increase in the number of samples analyzed in a single sequencing run. Prior to sequencing, the concentration of differently indexed samples was determined using a KAPA Library Quantification Complete kit . All indexed samples were diluted to 4 ng/μL, and 20 pM phiX DNA was added to a final concentration of 5% (v/v). Sequencing was performed using MiSeq Reagent Kit v3 (600 cycle) and MiSeq apparatus according to the manufacturer’s instructions . Quality trimming of the raw reads was performed using TrimmomaticPE v0.32 with a sliding window of 4 bp and a quality read score equal or higher than 20 [Caecal content samples were homogenized in a MagNALyzer . Following homogenization, the DNA was extracted using a QIAamp DNA Stool Mini Kit according to the manufacturer’s instructions . The DNA concentration was determined spectrophotometrically, and DNA samples diluted to 5 ng/mL were used as a template in PCR with forward primer 5′- than 20 . MinimallinA, tetA(P), tetQ, tetO, tet(32), tet(44), tetW, TEMbla, aadA, strA, and sul2 genes, as well as 10 selected microbiota members, including Anaerotruncus colihominis, Pseudoflavonifractor capillosus, Butyricicoccus pullicaecorum, Flavonifractor plautii, Olsenella uli, Bacteroides mediterraneensis, Bacteroides coprophilus, Mucispirillum schaedlerii, and Marseilla massiliensis. PCR was performed in 3μL volumes in 384-well microplates using QuantiTect SYBR Green PCR Master mix (Qiagen). Dispensing of the PCR master mix, primers, water and DNA was performed using a Nanodrop pipetting station . PCR and signal detection were performed with a LightCycler II (Roche) with an initial denaturation at 95 °C for 15 min followed by 40 cycles of denaturation at 95 °C for 20 s, primer annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Each sample was subjected to real-time PCR in triplicate and the mean values of the triplicates were used for subsequent analysis. Amplification of the 16S rRNA gene using Eubacteria specific primers was used as a reference to determine the total amount of bacterial DNA in each sample  and Proteobacteria . Higher abundance of Bacteroidetes and Proteobacteria occurred at the expense of Firmicutes, which were less abundant in outdoor hens  were selected as species representing genera more abundant in microbiota of outdoor chickens.In selected genera, their differential abundance was verified by quantitative real-time PCR. Pseudoflavonifractor capillosus and Olsenella uli as being more abundant in the microbiota of indoor chickens and for Bacteroides mediterraneensis, Bacteroides coprophilus, Mucispirillum schaedlerii, and Marseilla massiliensis as being more abundant in the microbiota of outdoor chickens. Real-time PCR did not confirm significant differences in abundance for Butyricicoccus pullicaecorum and Flavonifractor plautii, and conflicting results were recorded for Anaerotruncus colihominis. Although 16S rRNA sequencing predicted the genus Anaerotruncus as more abundant in indoor chickens, Anaerotruncus colihominis was significantly more represented in the microbiota of outdoor chickens , tetO, tetQ, tet(32), tet(44), tetW, and linA was determined. Of all tested antibiotic resistance genes, tetW, tetO, tet(32), and tet(44) were significantly more frequent in the microbiota of hens from commercial flocks than in the microbiota of outdoor, backyard hens. The difference in abundance of antibiotic resistance genes was therefore detected in genes commonly found in Firmicutes , tet(44), tetW) while the abundance of antibiotic resistance genes characteristic for Bacteroidetes (tetQ and linA) or Proteobacteria  was similar in both commercial and backyard hens , tet(44), and tetW genes characteristic of Firmicutes [Antibiotics are quite commonly used in commercial production while owners of backyard chickens rarely use antibiotics. This explains the lower abundance of antibiotic resistance genes in the microbiota of outdoor chickens. Similar data were reported also by Ferairo et al. . Due to roidetes  were thermicutes ,25,26 warmicutes ,27,28, armicutes ,30. HoweThe initial aim of this study was to identify microbiota members enriched in the microbiota of backyard hens since these may represent novel probiotics, which are for different reasons unavailable for commercially raised hens. However, when thinking critically, even the genera more abundant in the microbiota of commercial hens can be considered as having probiotic potential since their higher abundance in commercial hens indicates their higher adaptation for indoor hens’ genetics, feed, and living environment. Despite this, rather surprisingly, we realized that microbiota members that (i) were present at high but similar abundance in both indoor and outdoor hens but (ii) are usually absent from the microbiota of commercially hatched chickens during the first days of their life  and addi