Cephalotes) their stable symbiotic interactions with gut bacteria have persisted for 50 million years with the gut bacteria synthesizing essential amino acids that are used by the host. Although we know the function for some of these turtle ant-associated bacteria there are still many open questions.To understand the patterns of biodiversity it is important to consider symbiotic interactions as they can shape animal evolution. In several ant genera symbiotic interactions with microbial communities have been shown to have profound impacts for the host. For example, we know that for Camponotini the gut community can upgrade the host’s diet and is shaped by development and colony interactions. However, what is true for one ant group may not be true for another. For the microbial communities that have been examined across ants we see variation in the diversity, host factors that structure these communities, and the function these microbes provide for the host. In the herbivorous turtle ants  of more than 75 species of turtle ants across different geographic locations and in the context of the host’s phylogenetic history. Our results show (1) that belonging to a certain species and biogeographic regions are relevant to structuring the microbial community of turtle ants; (2) both bacterial and eukaryotic communities demonstrated correlations and cooccurrence within the ant host; (3) within the core bacterial community, Burkholderiaceae bacterial lineage were the only group that showed strong patterns of codiversification with the host, which is remarkable since the core bacterial community is stable and persistent.We concluded that for the turtle ants there is a diverse and evolutionarily stable core bacterial community, which leads to interesting questions about what microbial or host factors influence when these partner histories become evolutionarily intertwined.The online version contains supplementary material available at 10.1186/s42523-022-00223-7. To fully understand global biodiversity, we must focus studies on hyper-diverse groups such as arthropods, fungi, and microbes, which have a very large number of species and disproportionately fewer scientific studies compared to charismatic megafauna . TherefoCrematogaster [See Previous studies have shown that ants engage in various symbiotic interactions with fungi , 8, othester See .Pheidole [Atta sexdens [Still, ants are a great system for examining interactions with their microbial communities, as they are eusocial insects, thus providing an ideal scenario to facilitate bacterial transfer between individuals from the same colony . In addiPheidole  and Atta sexdens , 27. Stu sexdens , 11, 28) sexdens .Cephalotes is not fully known, it is believed that their diet is primarily herbivorous, including sap, pollen, insect honeydew and extrafloral nectar [To date, few studies have explicitly examined ant microbial communities and their potential for codiversification with their ant hosts , 30–33 al nectar , 36–38. l nectar –42. Thesl nectar , which cl nectar .Cephalotes species. However, with an increased scale of sampling, the inclusion of a host phylogenetic tree with greater resolution generated through a reduced representation genome sequencing approach [Previous studies have shown that turtle ant bacterial communities can be structured between different colonies within the same species , and alsapproach , and theCephalotes? (2) Are the bacterial and eukaryotic communities associated with turtle ants interacting? (3) Is the core bacterial community, which is stable and functional for the host codiversifying with the host when each bacterial lineage is analyzed independently? To achieve these goals, we used multiplex Illumina sequencing of 16S rRNA and 18S rRNA amplicons for more than 75 species of Cephalotes collected across the host’s geographic range. Our study provides new insights into this complex symbiotic interaction that turtle ants and their microbiomes have engaged for more than 50 million years.Therefore, with this study we intend to explore three questions in the diverse turtle ant microbiome: (1) What ecological factors structure the microbial communities of p value = 0.307) as previously found by Flynn et al. [p value = 0.322), so we kept samples from both in our library. After these initial filtrations and removing samples, our library contained 151 samples with 5,137,726 reads, varying with a maximum of 73,287 reads and a minimum of 2525 reads in the samples. Using BugBase, our data recovered more aerobic phenotypes than anaerobic in the 16S rRNA library  of more than 75 species of turtle ants across their geographic range and in the context of the host’s phylogenetic history. Our bacterial 16S rRNA preliminary results found differences between pinned museum samples using a noninvasive method of DNA extraction  and biogeographic regions  included in the present study , C. atratus and C. unimaculatus (both increasing from average), appear to be driving much of this difference, and for biogeographic regions our results indicate that the Antilles (increasing from average), is impacting this result. All results from these specific groupings can be found highlighted in Additional file Bacterial quantification (16S rRNA) was performed by qPCR and our results found significant differences between the bacterial communities for the host species , 2% Lactobacillaceae (Lactobacillus), and others in smaller abundance  was composed of 29% Burkholderiaceae, 28% Xanthomonadaceae, 26% Rhizobiaceae, followed by 7% Opitutaceae, 2% Rickettsiaceae 6S rRNA wCephalotes with Shannon and Simpson metrics . Still, when we explore these pairwise results, we see that only a few combinations of species are driving these results. Those species that showed significant results (< 0.05) are highlighted in Additional file p value = 0.518).Overall, our data show high bacterial alpha diversity within  workers , 49–51. To explore these results more deeply, we investigated whether any core bacteria exhibit specificity to a particular species or biogeographic region, or whether the distribution of ASVs is more widespread. Our results presented in Additional file p value = 0.254) or in biogeographic region-based comparisons . We also explored if any microbial eukaryotes exhibit specificity to a particular species or biogeographic region. However due to the low taxonomic resolution of the 18S rRNA database, a large part of our data was removed (those who were only identifed as Eukaryote). Therefore, we cannot thoroughly investigate this question  and different biogeographic regions . The unweighted unifrac distance results can be seen in Fig. Cephalotes samples present in this study and found both categories can structure bacterial communities  and abundance (unweighted unifrac distance) in both bacterial (16S rRNA) and eukaryotic (18S rRNA) communities Fig.  of turtlp value = 0.397, weighted unifrac, Pseudo-F = 1.121, p value = 0.347). Also, when we explore the different biogeographic regions, the same pattern was identified with no impact on both composition and abundance   host species has no impact on both composition and abundance . We see that for the groupings of species, both the composition and the abundance of bacterial communities seems to be significant overall for species C. grandinosus and C. pusillus, but considering only the composition, we find overall differences between C. atratus and C. maculatus  between 16S rRNA and 18S rRNA ASVs associated with the Cephalotes samples, with 172 interactions identified as positive correlations and 187 as negative correlations as can be seen through the network structure in Fig. Our analysis revealed 359 significant correlations  analyses  for the genus phalotes . Each coses Fig. . Comparieae Fig. , while tInvestigating host-associated microbial communities can reveal insights into the ecological and evolutionary success of the host , 54. InsWithin the Formicidae ant family it is no different and growing evidence suggests that the partnership with microbes was fundamental for ants to reach new niches , 4. ManyWolbachia. This pattern of conserved bacterial community has already been observed by other insects such as the social bee [Blochmannia interactions, the core bacterial community of Cephalotes provides fundamental functions for the host by contributing to nutritional upgrading with nitrogen recycling and synthesis of essential and non-essential amino acids [The core bacterial community results from this study corroborate previous work on turtle ants, emphasizing that associated bacteria are highly conserved , 33–35. cial bee  and alsocial bee , 50, 63.no acids , which aCephalotes samples are involved in upgrading the diet by recycling urea and obtaining nitrogen for the host [Cephalotes and some of them have already been identified in other species of ants as well, such as Rhizobiales. This bacterial group has been associated with other herbivorous species such as Dolichoderus [Hapergnathos saltator, Pheidole, Paraponera clavata, Daceton armigerum and army ants [Evidence from previous studies suggests that Burkholderiales, Opitutales, Rhizobiales and Xanthomonadales found in the host , 33, 35 the host . But thechoderus , but alsrmy ants , 65, 66.rmy ants .Cephalotes samples in previous works was Opitutales, which was composed almost exclusively of the genus Cephaloticoccus, previously found in Cephalotes [Cephalotes samples suggesting that it may assist in the acquisition of nitrogen for the host [Another order of bacteria identified in this study that was strongly associated with phalotes , 44, 68.the host . Interesthe host . AlthougSpecifically in ants, several studies have already showed that there is structuring of bacterial communities of different species, stage of development within the colony, parts of the body and different tissues, habitats, host phylogeny and even the diet of the host, 49, 50.Cephalotes bacterial communities in different gut chambers [Cephalotes varians [Cephalotes. In addition, species and biogeographical region of the ant host impacted bacterial quantification (copies of 16S rRNA) identified through qPCR. We also found significant differences for the abundance of bacterial communities within the different species of Cephalotes analyzed. In addition, we found differences in abundance of bacterial communities in the different biogeographic regions, but the same pattern was not observed for eukaryotic communities.In the case of turtle ants, despite having a stable bacterial core community , 34, 35 chambers , 33, 44,Cephalotes hosts. In general, if these interactions of eukaryotic and bacterial communities show mutualism, parasitism, or a tripartite interaction (or more parts) it still deserves to be further explored in future studies. As one of the examples, Wolbachia showed positive and negative interactions with several different microbial eukaryotic taxa. This bacteria is one of the most well-known bacteria infecting insects, including social insects [Wolbachia and these other taxa are undescribed and deserves to be explored in depth.Interactions between microbial communities and hosts are being increasingly studied, including eukaryotic and bacterial domains , 53. Our insects  whose co insects , 72). ThBuchnera and aphids [Snodgrassella and Gilliamella bacteria in corbiculate bees [Blochmannia and Camponotini ants [Cephalotes, suggesting that this pattern may reflect codiversification of core bacteria and host or a similar selective environment between related hosts [Some symbionts have shown patterns of codiversification with the host, probably facilitated by a high fidelity to vertical transmission, and this is evidenced by the congruence of the host’s phylogenetic tree and the symbiont as already observed in d aphids , Snodgraate bees –76, Blocini ants  and Firmini ants . Previoued hosts .Cephalotes were able to find correlation between the bacterial community and the phylogeny of the host [Cephalotes species shows that for some lineages we failed to find evidence of codiversification with the host turtle ants, and we only found Burkholderiaceae bacterial lineage are codiversifying and being consistently transmitted vertically or may have varying histories of the timing of acquisition or frequent horizontal gene transfer between closely related bacteria. Another suggestion is that some of these bacteria are being picked up from the environment, which may explain the signal from the biogeographic regions structuring the microbial communities in this present study. This same trend was observed in deep-sea anglerfish, despite different species sharing the main symbionts, these are not acquired vertically. In this system, deep-sea anglerfish acquire the symbionts from the environment in every new generation [Approaches that take individual host-associated bacterial groups separately into account should also be explored. For example a study conducted recently with the abdomen/gaster of eleven species of the host , a resulneration .Horizontal acquisition of beneficial microbes through anal trophallaxis is essential in colonies of bees and termites –80. The Cephalotes hosts, which leads to interesting questions about what microbial or host factors influence when these partner histories become evolutionarily intertwined.Overall, we have shown that for the turtle ants there is a diverse and evolutionarily stable core bacterial community that is structured by host species and to some extent biogeography of the host. Although we found some signal of microbial interactions between bacteria and microbial eukaryotes the lack of a highly populated microbial 18S rRNA database precluded us from fully exploring these data. We did find codiversification of Burkholderiaceae bacterial lineage with their Cephalotes, representing more than 75 different species and covering the entire geographic distribution of the genus. The complete list of specimens and location can be found in Additional file For this study we included 192 samples of http://www.earthmicrobiome.org/protocols-and-standards/). For each sample three PCRs were performed containing 12 μl of PCR water (Certified DNA-free), 10 μl of 5 Prime HotMasterMix (1 ×) , 1 μl of forward primer , 1 μl of reverse primer  and 1 μL of template DNA (> 0.20 ng/μl), totaling a final volume of 25 μl per PCR reaction. These reactions were placed in the thermocycler under the following conditions: 94 °C for 3 min, with 35 cycles at 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s, with a final cycle of 10 min at 72 °C. Agarose gel electrophoresis (1%) confirmed the efficiency of the amplification. Quantification by Qubit (Thermo Fisher Scientific) was performed with the High Sensitivity Assay Kit . Quantification by qPCR was also performed (see description below). Afterwards, samples were pooled to a total of 100 μL per pool, and purified with QIAquick PCR Purification Kit , following the manufacturer's recommendations. Each pool was diluted to 4 nM and then denatured. Then the pool was further diluted to a final concentration of to 6.75 pM with a 10% PhiX, following Illumina recommendations. Sequencing was performed at Argonne National Laboratory  with two separate runs (16S and 18S rRNA) through MiSeq Illumina V3 Reagent Kit 600 Cycles (300 × 300) using the custom sequencing primers and procedures described in the supplementary methods in Caporaso et al. [DNA extraction used DNeasy Blood & Tissue Kits  and DNeasy PowerSoil Kit  with the modification of a beat-beating step and addition of Proteinase K and was performed according to the manufacturer's recommendations. Total DNA was extracted from either from one gaster (abdomen) or one whole worker, or even in the case of material deposited in collections with the non-destructive methodology. Subsequent analyses controlled for these variations so as not to affect the results. Additionally, recommendations by Rubin et al.  and Moreo et al. . All rawhttp://earthmicrobiome.org/emp-standard-protocols/16s/) to target the bacterial 16S rRNA gene using a CFX Connect qPCR machine , with SYBRAdvanced 2X (Bio-Rad) SYBR green supermix and 2 μL of DNA. All samples were quantified in triplicate. Serial dilutions of plasmids containing inserts of E. coli 16S rRNA were performed to establish standard curves [Quantification of the abundance of bacteria was performed with real-time qPCR using the universal primers 515f (5′-GTGCCAGCMGCCGCGGTAA) and 806r (5′-GGACTACHVGGGTWTCTAAT)  tests were also implemented in QIIME2. To test whether variations in DNA extraction kits or different sample types  impacted our results, PERMANOVA analyses were conducted, and our results show that we only found differences between non-destructive samples. Therefore, these were excluded from our subsequent analyses, with 151 samples remaining.The microbial phylogeny was performed using SATé-enabled phylogenetic placement (SEPP) , 100. We package  in the e package  to predindations  and werendations  with 200Subsequent analyses tested the influence of both: (I) host species identity and (II) geographical distribution of the host turtle ants on both microbial communities. For the latter, we followed the recommendation of Price et al. , and divTo test the potential for correlation of the 16S rRNA and 18S rRNA libraries obtained in the present study we performed a Regularized Generalized Canonical Correlation Analysis (rGCCA) through the mixOmics package , 109 in Cephalotes phylogenomic tree of Price et al. [Each microbial lineage has an independent and complex evolutionary history with the host, therefore two methods were applied to test for coevolutionary signal between the core gut bacteria (50%) and the host’s evolutionary history: Mantel test and PACo (Procrustean Approach to Cophylogeny) analyses , 112. Foe et al.  was usede et al.  in R. The et al. , 112. FoFor a better visualization of host-bacterial associations, tanglegrams were plotted with the Phytools package  in the RAdditional file 1. Sample type differences between samples of Cephalotes used in this study. Note that there is no difference between DNA extract kits and the abdomen/gaster and the whole workerAdditional file 2. Aerobic and anaerobic phenotype bacteria identify in turtle ants. BugBase results for 16S rRNA in Cephalotes in the present study. Additional file 3. Bacterial quantification (qPCR) through the 16S rRNA gene (515F/806R) associated Cephalotes samples. Note that belonging to a species and a biogeographic region impact the results of the quantity of bacteria (number of copies of 16S rRNA) in the host. Additional file 4. Species and biogeographic regions that impact qPCR results. Group 1 consists of the average of the entire dataset to be compared with group 2. Those that presented significant results are highlighted in red.Additional file 5. Alpha diversity found in samples of Cephalotes measured by Shannon and Simpson indices. A High alpha diversity found in bacterial communities associated with turtle ants. B Alpha diversity in eukaryotic microbials associated with turtle ants is lower compared to the bacterial community. Note included in these visualizations alpha diversity can be compared for different species as well as by different biogeographic regions.Additional file 6. Kruskal–Wallis pairwise tests of combinations of species of Cephalotes that showed significant results.Additional file 7. Bar plots illustrating the abundance of the most common bacterial orders associated with turtle ants. A Main orders of ASVs are grouped according to different host species of Cephalotes. B Main orders of ASVs grouped according to different biogeographic regions of Cephalotes distributionAdditional file 8. Bar plots illustrating the abundance of most common Eukaryotic orders associated with turtle ants. A Main orders of ASVs grouped according to different host species of Cephalotes. B Main orders of ASVs grouped according to different biogeographic regions of Cephalotes distribution.Additional file 9. Non-metric multidimensional scaling (NMDS) (Bray-Curtis) ordination of bacterial communities of Cephalotes samples colored according to different species and biogeographic regions with 95% confidence intervalAdditional file 10. List of specimens included in this study.


Assessment of immune competence of farmed Atlantic salmon is especially important during smoltification and the first several months in the sea. Recently developed tools were applied to salmon raised in a traditional flow-through facility  and in a recirculation aquaculture system . Fish were sampled at four time-points: parr, smolt, and at three weeks and three months after seawater transfer (SWT); expression of 85 selected immune and stress genes, IgM transcripts (Ig-seq), and circulating antibodies were analyzed. A steady increase in gene expression was seen over time in gill and spleen in both cohorts, and especially in antiviral and inflammatory genes in the gill. Differences between the cohorts were greatest in the dorsal fin but later leveled off. Comparison with a gill reference dataset found a deviation in only three of 85 fish, suggesting a good immune status in both cohorts. Levels of both specific and nonspecific antibodies were higher in cohort 2 in smolts and in growers three weeks after SWT; however, levels evened out after three months in the sea. Ig-seq indicated association between antibody production, expansion of the largest clonotypes, and massive migration of B cells from spleen to gill in smolts. The results suggested greater agitation and higher reactivity of the immune system in RAS-produced salmon, but the difference between the cohorts leveled off over time. Atlantic salmon aquaculture is undergoing rapid development, and new technologies are being introduced to address issues of fish health and welfare as well as environmental concerns. Flow-through (FT) aquaculture systems belong to the traditional production systems, whereas recirculation aquaculture systems (RAS), which reduce the consumption of water and prolong the rearing of fish in land-based facilities, are among the main innovations, and their share in smolt production is rapidly increasing ,2,3. As The immune system of Atlantic salmon is influenced by a combination of factors including production protocols, farming conditions, developmental changes, and infection pressure. The occurrence of diseases is greatest after seawater transfer and during the first several months in the sea ,7. HigheThis case study assessed the immune status of two cohorts of Atlantic salmon produced at a traditional FT facility and a RAS facility within the period from parr to three months after seawater transfer (SWT), using recently developed tools. The multigene expression assay (MGE) is narrowing a gap between qPCR analyses of small sets of immune genes that has proven insufficient for diagnostics and high-throughput methods (“omics”) not suitable for large numbers of samples due to high costs, limited capacity, and complexity of data management. We developed an MGE assay for the assessment of immune competence of Atlantic salmon (ImCom) on the Biomark HD platform. Immune and stress genes were selected from Nofima’s transcriptome database according to the scale and reproducibility of responses to pathogens . ImCom hMultiplexed antibody assays allow comparison of responses to various antigens in the same sample. In addition to assessment of specific antibody responses to antigens delivered with vaccines, we included nonspecific antibodies (NSAB). NSAB can be detected in the assay by binding circulating antibodies to model antigens toward which the Atlantic salmon has never been exposed. Our recent studies showed strong responses of NSAB to vaccination, functional disorders and infections with pathogens indicating their importance in Atlantic salmon ,14,15,16The reported study assessed these tools on field material: Atlantic salmon reared in commercial aquaculture.Two cohorts of Atlantic salmon reared in Southwestern Norway were examined . Fish maFish in cohort 2 were kept in a recirculation aquaculture system (RAS) with >95% recirculation for the final 11 weeks before SWT. Oxygen levels were measured daily and maintained at >90%. Fish were fed a commercial freshwater feed . The production unit had an average water temperature of 13.5 °C. A 12:12 photoperiod regime was applied up until vaccination and for 5 weeks after vaccination, followed by 24:00 regime for the final 5 weeks until SWT, which took place at an average weight of 113.85 (+/−24.8). Fish were vaccinated i.p. with commercial vaccines  at an average weight of 50.5 g. Fish health and performance and facility production parameters were regularly monitored by on-site staff and fish-health veterinarians.Sampling regimes are included in In cohort 2 (RAS), samples were collected shortly before vaccination  and before SWT at 754 DD after vaccination . Fish condition factor and smolt index were assessed as above. Fish were further sampled at 3 weeks after SWT  and at approximately 3 months after SWT .For lethal sampling, fish were anaesthetized with tricaine  and killed with a blow to head. Organ samples including gill (two filaments from first left gill arch), dorsal fin , and spleen were placed in RNALater  and stored overnight at 4 °C before further storage at −80 °C. Blood was collected from the caudal vein with a vacutainer containing EDTA anticoagulant (VWR), and plasma was immediately separated with centrifugation before further storage at −80 °C.Small pieces of dorsal fin, gill, and spleen (5–10 mg) were placed in tubes with 400 µL lysis buffer  and beads, and 20 µL proteinase K (50 mg/mL) was added in each tube. Samples were homogenized in FastPrep 96  for 120 s at maximum shaking, centrifuged, and incubated at 37 °C for 30 min. RNA was extracted on Biomek 4000 robot using Agencourt RNAdvance Tissue kit according to the manufacturer’s instructions. RNA concentration was measured with NanoDrop One , and quality was assessed with Agilent Bioanalyzer 2100.−4.Analyses were performed in spleen and gill as described in . SynthesTenacibaculum) pathogens of key importance for Atlantic salmon aquaculture (ef1f and rps20), which showed stability across samples, were used for calculation of ΔCt, and ΔΔCt was calculated by subtracting the average ΔCt from each value. Differential expression of individual genes was assessed by criteria applied to microarray data: difference of ΔΔCt > |0.8| and p < 0.05. Gene expression in the gills was compared to a reference dataset (GRDS) with 595 healthy salmon covering different ages, seasons, areas of Norway, and aquaculture systems. Differences from the mean ΔΔCt values for each gene in GRDS were averaged in the samples. The deviation from GRDS for 2σ was set as the threshold value for outliers.The selection of genes, design, and validation of the assay are described in ,16. Seveaculture . The genAeromonas (A.) salmonicida subsp. salmonicida ([Moritella (M.) viscosa type strain 1016/96 were included in the multiplex assay for the detection of antibodies induced with vaccination. DNP-keyhole limpet hemocyanin DNP-KLH  was used for the detection of nonspecific antibodies (NSAB). Antigens were coupled to distinct MagPlex®-C Microspheres  of different bead regions according to the manufacturer’s protocol using the Bio-Plex amine coupling kit . DNP-KLH was used at 10 µg per 1× scale coupling reaction, and A-layer protein and M. viscosa sonicate at an amount of 12 µg and 7 µg, respectively. Briefly, beads were diluted in assay buffer (PBS with 0.5% BSA and 0.05% azide), and 5000 beads per region were added to each well. Beads were washed three times with assay buffer (30 s in the dark and on a shaker at 800 rpm), then kept for 120 s in a Bio-Plex handheld magnetic washer before the supernatant was poured off. Following initial titrations, plasma samples were diluted 1/4800 in assay buffer and added in duplicates on the plate, except for samples from parr (1st sampling) which were diluted 1/1200 in assay buffer. The plate was incubated for 30 min at RT in the dark and on a shaker at 800 rpm. All subsequent incubation and washing steps were performed similarly. Following incubation and washing, Anti Salmonid-Ig (H chain) monoclonal antibody  was added in each well. After incubation and washing, biotinylated goat Anti-Mouse IgG2a antibody  was added in each well, and finally, after incubation and washing, Streptavidin-PE . Each bead was classified by its signature fluorescent pattern and then analyzed for the median fluorescence intensity (MFI) of the reporter molecule.Analyses were performed as described previously . The A-lonicida  in plasma, as previously described . All solComparison of data within each cohort was analyzed with ANOVA followed with Tukey or LSD post-hoc test using Statistica 13.One week before SWT, an average condition factor and smolt index of respectively 1.25 and 3.15 were recorded in fish from cohort 1 with an average weight of 87.4 g (+/−12.5 g). An average weekly site total mortality of 0.03% was recorded during the first 3 weeks after SWT, and the recorded feed conversion ratio (bFCR) was 1.0 during the same time period. After approximately 3 months in the sea, the average fish weight was 726 g, and the site total mortality and the bFCR were 0.04% and 1.0, respectively.In cohort 2, an average condition factor of 1.27 and a smolt index of 3.80 were recorded the week before transfer in 113.85 g (+/−24.8 g) fish. An average weekly site total mortality of 0.02% and bFCR of 1.0 was recorded during the first 3 weeks after SWT. After approximately 3 months in the sea the average weight was 668.8 g, the site total mortality was 0.02% and the bFCR 1.0.In addition to immune and stress genes, MGE assay included primers to selected viral and bacterial pathogens of major concern for Atlantic salmon aquaculture. Fish were negative for infectious salmon anemia (ISAV), infectious pancreatic necrosis (IPNV), and piscine myocarditis (PMCV) viruses in fin, gill and spleen at all sampling time-points. Piscine orthoreovirus (PRV) and Salmonid alphavirus (SAV) were detected in respectively 14 and 4 (sea only) fish from a total of 85 analyzed fish from both cohorts . TenacibMultigene expression assay was performed on three tissues: dorsal fin, gill, and spleen. Mean ΔΔCt is a simple metric reflecting overall expression levels of all immune genes included in the assay . The larComparison of immune gene expression in the gill with the gill reference data set (GRDS)  by ΔΔCt A. salmonicida and NSAB) or further increase (M. viscosa) after 3 weeks in the sea and a steep decrease at the last time-point. Antibody levels in cohort 1 (FT) increased steadily throughout the sampling period but were at a lower level than in cohort 2 in smolts and after 3 weeks in the sea and until the last sampling point, when the antibody levels evened out between the two cohorts. The lower antibody levels in fish from cohort 1 were also reflected in the total plasma immunoglobulin levels (M. viscosa) than between the two vaccine-specific antibodies (r = 0.61) (not shown).The kinetics of specific antibodies recognizing vaccine components and nonspecific antibodies binding to a model antigen were analyzed by multiplex immunoassay . In cohon levels . The corSequencing of the IgM repertoire (Ig-seq) was performed to explore possible links between the kinetics of circulating antibodies and expansion of antibody-producing cells. Mounting of acquired immunity is associated with enhanced B-cell proliferation and IgM transcription within clones producing antibodies to recently delivered antigens, and both processes increase absolute (counts) and relative (frequencies) amounts of transcripts. Transcripts from newly expanded clones appear among the largest clonotypes, thus increasing the cumulative frequency (CF) of transcripts from the leaders. Here, CF of the fifty largest clonotypes (CF50) significantly increased in the gills of salmon from both cohorts and stabilized at high levels A,B. SimiUnique immunoglobulin sequences or clonotypes mark B cells of clonal origin. The co-occurrence of identical clonotypes in the spleen and gill reflects the migration of B cells from the lymphoid (spleen) to the peripheral organ (gill). In Atlantic salmon, sharing of clonotypes between organs is relatively low under basal conditions but increases after strong immunization, followed by a gradual decrease because the largest clonotypes in the lymphoid organ are substituted with recently expanded clones. Recruitment of B cells to gills was markedly stimulated in smolts of both cohorts A,B, as iControlled experimental trials contribute essential knowledge on the immune system of Atlantic salmon; however, they do not necessarily reflect the complexity of immune responses in fish in commercial production. Until present, fish immunology has focused mainly on responses to pathogens, vaccination, stress, and other disturbances, while much less is known about the immune status of healthy fish in farming conditions. This descriptive case study is a step toward the development of methods for the assessment of the immune competence of farmed Atlantic salmon. The objective was twofold: (1) testing of newly developed tools on field material from commercial Atlantic salmon aquaculture and (2) assessment of the immune status of fish reared in different aquaculture systems.The multigene expression assay (MGE) has been applied to material from controlled experiments and smolt batches with contrasting health conditions ,16,20. HParticular attention was paid to two diagnostic gene sets. An unexpected finding was a significantly higher abundance of VRG transcripts in the dorsal fin of cohort 2 (RAS) at first (parr) and third (3 weeks post-SWT) samplings. This was, however, not observed in the gills and spleen, and the detected increase in VRG in the fin was much smaller than what was previously observed in virus-infected Atlantic salmon , suggestp = 0.05) is not a pathology per se but can be considered as a warning sign. In the present study, with a normal distribution of the metric, the observed number of fish beyond the threshold of the GRDS was low, indicating a robustness of the external reference data set. No fish exceeded the 2σ threshold, and the mean expression of this set was 4.2 and 15.6 times lower than that of previously analyzed smolt batches with high mortality , is a crucial step in the development of tools for assessment of immune status of Atlantic salmon. Currently, to determine the boundaries of the norm, we can only rely on statistical parameters—a deviation of more than 2σ  in smolts and in growers 3 weeks post-SWT; however, levels evened out between cohorts after three months in the sea. The difference in antibody levels in smolts between the cohorts is most likely explained by the difference in number of degree-days between the cohorts from vaccination to sampling of smolts, but it may also be influenced by the vaccines, the farm production protocol, or their combination. The persistently high levels of circulating NSAB in Atlantic salmon are in stark contrast to the almost complete lack of knowledge about their role. Moreover, strong correlation between levels of vaccine-specific and nonspecific antibodies is intriguing and is evidence in favor of diagnostic usefulness of NSAB, especially for multicomponent vaccines.Cyprinus carpio) to a panel of human proteins after challenge with herpesvirus [Vaccination may equally stimulate different populations of B cells, including those recognizing vaccine-delivered antigens and bystanders producing antibodies of broad reactivity. An alternative explanation is that the vaccine and model antigens, such as DNP-KLH, are recognized by the same antibodies. These antibodies may display a broad reactivity but most likely exhibit a lower affinity. This latter possibility is supported with markedly enhanced nonspecific binding of IgM in sera of carp  and increased sharing of clonotypes between a peripheral organ (gill) and a lymphoid organ (spleen), which was highest in smolts (cohort 2) and after three months in the sea (cohort 1). We have reported enhanced traffic of B cells from the lymphatic organs to peripheral tissues and indicated its protective role in fish infected with salmonid alphavirus . FollowiIn the present case study, tools developed for assessment of immune competence of Atlantic salmon were validated on field material from commercial aquaculture. Comparison of gene expression with an external gill reference data set suggested good immune status of fish reared in both FTS and RAS, as well as a robustness of the external reference data set. Differences between the cohorts that were observed before seawater transfer levelled out after several months in the sea. Future development of diagnostics requires defining the boundaries of normality as well as their biological significance.


Zingiber officinale). Despite its nutritional value, it is difficult to preserve or further process and thus is often wasted. This study uses spent ginger as a substrate for fermentation and cultivates spent ginger yeast cultures (SGYCs) that are then added to the feed of laying hens. The effects of SGYCs on production performance, egg quality, serum composition, and intestinal microbiota of laying hens were investigated.Spent ginger is a byproduct of juice extraction from the rhizome of ginger . The control group was fed a basal diet while experimental groups were also given SGYCs at the levels of 5, 10, 20, and 40 g/kg for 6 weeks.The addition of SGYCs significantly increased the laying rate and nutrient digestibility, decreased feed conversion ratio, and enhanced the color of egg yolks (p<0.05). No changes were observed in activity levels of alanine aminotransferase and aspartate aminotransferase in the serum (p>0.05), but the activities of superoxide dismutase, glutathione peroxidase, and peroxidase all significantly increased, and contents of malondialdehyde were significantly reduced (p<0.05). In addition, changes in the relative abundance of Firmicutes and Bacteroidetes might be the main factor contributing to the significant increase in the apparent digestibility of crude protein and crude fat in laying hens (p<0.05).The current evidence shows that dietary supplementation of SGYCs to the feed of laying hens can improve laying rates, enhance antioxidative defenses, and influence dominant intestinal bacteria. Eggs are considered an ideal food for humans in terms of nutritional value. Egg protein has amino acid composition highly suitable and can be easily absorbed by the human body, with utilization rates up to 98% . Eggs arSaccharomyces cerevisiae [S. cerevisiae]) and the solid or liquid mediums in which they were fermented, are considered high-quality supplements because they contain various bioactive substances including yeast cells, vitamins, amino acids, organic acids, and oligosaccharides [Supplementation to the diet of laying hens is a common strategy to improve egg produc tion. Yeast cultures (YCs), which typically refer to mixtures of both yeast  to extract juices. Due to the high moisture content in spent ginger, it is difficult to preserve or further process, and thus often discarded. However, it is also an ideal substrate to culture YCs for feed supplementation. Large molecules such as proteins and starches in spent ginger can be broken down into by yeast, which is conducive to absorption. As well, ginger contains various monoterpenes , sesquiterpenes , phenols  to the diet of laying hens may affect their blood indicators and intestinal bacteria, as well as their productivity and the quality of eggs produced. We also explore the potential mechanisms through which SGYCs influence the output and quality of egg production. Our work aims to reduce the waste of ginger byproducts and promote the sustainable development of animal husbandry.The present experiment was reviewed and approved by the Institutional Animal Care and Use Committee at Shanghai Academy of Agricultural Sciences (SAASPZ0920001).S. cerevisiae  at 0.4 g/L. The mixture was fermented in a semi-solid state at 20°C until sugar contents were less than 4 g/L and no longer changed for two consecutive days, then squeezed with a plate and frame press. The pulp was dried at 55°C and crushed. The proximate composition of SGYCs was as follows: 9.6% moisture, 9.8% crude protein, 5.2% crude fat, 24.5% crude fiber, 6.0% ash, and 45.0% total crude carbohydrates.Hy-Line Brown laying hens (60 weeks of age) were provided by Jun’an Egg Farm . Spent ginger yeast cultures were provided by the Fermentation Engineering Laboratory of Shandong Agricultural University . Cultivation procedures were as follows: spent ginger was mixed with water at a 1:3 ratio, to which sucrose was added at 102 g/L and A total of 80 Hy-Line Brown laying hens were randomly allocated to one of five treatment groups. Each treatment had 4 replicates with 4 hens per replicate. The basal diet of the five treatment groups were supplemented with SGYCs at levels of 0 (control), 5, 10, 20, and 40 g/kg. All experimental diets were approximately isonitrogenous and isocaloric and were formulated to meet or exceed nutrient requirements recommended by the NRC . IngrediThe study was conducted for 8 weeks, including 2 weeks of adaptation and 6 weeks of the experiments. The laying hens were housed in an environmentally controlled room where the temperature was 20°C±2°C. A 16-h lighting schedule (16 light:8 dark) was used during the entire experiment. Hens were raised in cages: each cage was 400 mm long, 450 mm deep, 450 mm high in the front and 380 mm high in the rear. The bottom of the cage leaned 7 degrees and was equipped with a nipple drinker and a trough feeder. Each cage had 4 laying hens, which could eat and drink freely. Feeding, egg collection, and cleaning of the coop were performed daily. Feces were cleaned and the chicken coop was sprayed and disinfected once a week.During the feeding trial, the total number of eggs and total egg weight of each replicate were recorded every day. Feed consumption of each replicate was recorded each week. The average egg weight (g), laying rate (%), average daily feed intake (g), and feed conversion ratio of each replicate were calculated at 3-week intervals. The average egg weight was the ratio of the total number of eggs to the total egg weight. The laying rate refers to the number of hens that laid eggs as a percentage of the total number of laying hens. The feed conversion ratio was calculated by dividing the weight of feed consumed by the weight of eggs produced.To determine egg quality, 12 eggs were randomly selected from each group on day 21 and day 42 of the feeding experiment. The major and minor axes of the eggs were measured using the egg shape index meter , and the egg shape index (long diameter/short diameter of eggs) was calculated. An egg quality tester  was used to determine eggshell hardness (kgf), yolk color, albumen height (mm) and the Haugh unit. The determination of yolk color value was based on the “Roche color fan (RCF)” system, which consists of 15 color samples corresponding to values 1 to 15. The thickness of the shell at the blunt end, tip and middle of the egg was measured using an eggshell thickness tester  and used to calculate average eggshell thickness.Apparent digestibility was determined using the total feces collection method . After tOn the 42nd day of the experimental period, 12 laying hens were randomly selected from each treatment group of 16. Blood samples were collected from the wing vein of each hen using disposable needles, and then placed in a vacuum sealed sampling vessel. A blood cell analyzer  was used to determine routine indices in the whole blood, including red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV) and platelet count (PLT). Blood biochemical indicators were determined using the serum. Blood samples were centrifuged  at 4°C in a centrifuge  to obtain serum samples, which were stored at −80°C. The following biochemical indicators were determined using an automatic biochemical analyzer : glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), and total cholesterol (CHOL).The serum samples prepared in the previous section were used to determine the serum antioxidant capacity. The contents of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), peroxidase (CAT), and malondialdehyde (MDA) in the serum were determined using a microplate reader  and a Beyotime Biotechnology testing kit , following manufacturer’s instructions.On the 42nd day of the experimental period, 4 laying hens were randomly selected from each group to collect fresh feces. The fresh feces were placed in a sterile cryotube and placed in liquid nitrogen for rapid freezing, then sent to the laboratory to be stored in at −80°C for subsequent analyses. Fecal samples were separately iced and homogenized, and each was processed using a QIAamp DNA Stool Mini Kit  in strict accordance with the manufacturer’s standard procedures to extract microbial genomic DNA. Primers 338F (5′-ACTCCTACGGGAGCAG CA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT -3′) were used for polymerase chain reaction amplification to detect V3–V4 regions of bacterial 16S rRNA. Sequencing and bioinformatics analysis were performed by PersonalBio Ltd. . Briefly, paired-end sequencing of community DNA fragments was conducted on the Illumina Miseq platform, and the original sequencing data was saved in FASTQ format. Sequences were quality filtered, denoised, merged, and had chimeras removed using the DADA2 plugin . SequencStatistical analyses were performed using SAS 9.2. A one-way analysis of variance was performed to detect significance differences among groups. Means were also compared using Tukey’s multiple comparison test to determine pair-wise differences. The linear and quadratic responses were analyzed using orthogonal polynomials. Differences at p<0.05 were considered significant, whereas p-values between 0.05 and 0.1 were interpreted as trends. The Spearman rank correlation coefficient calculated in IBM SPSS Statistics 25 was used to analyze correlations, with p<0.05 indicating significant correlation and p<0.01 indicating extremely significant correlation.S. cerevisiae, and found that increased egg-laying rate and reduced feed conversion ratio when a 0.3% supplementation of S. cerevisiae cultures was added to feeds.As seen in 40H56O2). Common pigments present in feeds include lutein, zeaxanthin, and cryptoxanthin [21H20O6) found in ginger rhizomes. Curcumin is an isomer of lutein with very similar properties. Park et al [Although SGYC supplementation did not significantly change the properties of the eggshell including shell strength, shell thickness, and shape index, it had a significant effect on the yolk color. Analysis of eggs collected on the 42nd day of the experimental period showed that yolks of eggs laid by hens from the treatment groups were significantly different in color compared to the control (p<0.01), and the RCF values of yolk color obtained from the 40 g/kg group were as high as 8.90. The color of egg yolks is known to be a key factor in the marketing and sales of eggs, and golden or orange yolks with RCF>8.0 are typically more preferred by consumers . Researcoxanthin . The enhrk et al  have alsS. cerevisiae cultures to the diet of laying hens can increase activities of digestive enzymes in the chyme while also decreasing the toxin levels in their plasma.The apparent digestibility of nutrients in laying hens are as shown in In addition, results of the present study show that during the latter half of the experimental period (day 21 to 42), the laying rate was significantly positively correlated with the apparent digestibility of crude fat, crude fiber, crude protein, and ash (p<0.05). Therefore, we conclude that the apparent digestibility of nutrients is closely related to the laying rate. Other studies have also shown that increasing the digestibility of nutrients in laying hens can improve rates of egg production .Alterations in hematological indices including the decrease of RBC, HGB, and HCT are the main symptoms of anemia . As showAs nutrients enter the blood after being absorbed and converted, the increased digestibility of nutrients in hens fed SGYCs is also reflected in nutrient levels in the serum. The results revealed that TP and CHOL content in the serum were significantly positively correlated with the apparent digestibility of proteins and fats, respectively (p<0.01). Higher protein content in the serum is in turn correlated with egg albumen height (p<0.05). As albumen height is a main indicator in evaluating egg quality that reflects both nutrient levels and egg white consistency, the results also corroborate previous research that serum protein content is a key factor in determining egg quality .2•−), which are cytotoxic free radicals that can attack the polyunsaturated fatty acids in the biofilm, causing lipid oxidations and generating MDA [2•− [As a byproduct of their metabolism, laying hens produce superoxide anions (Oting MDA . MDA wilMDA [2•− . Thus, tAs seen in The intestinal microbiota community structure is shown in The current evidence shows that dietary supplementation of SGYCs to the feed of laying hens can improve laying rates and decrease feed conversion ratio, and such effects are the most prominent when they are added at 10 g/kg of feed. Eggs laid by treatment group hens also had yolks with enhanced color. Additionally, enzymatic activity of antioxidants in the serum also significantly increased, suggesting a lower occurrence of physiological abnormalities. These findings all support the potential application of SGYCs as a feed supplement that benefits laying hens. The potential mechanisms through which SGYCs influence production performance and egg quality of laying hens are summarized in


Late-gestation heat stress of dairy cows reduces fetal growth and influences postnatal performance and immune status of the offspring. Our first objective was to evaluate the effect of in utero heat stress on overall fetal and organ growth, particularly organs associated with immune function. The second objective was to examine the cellular mechanism of altered passive immunity in neonatal bull calves after in utero heat stress. Specifically, we examined the rate of apoptosis of intestinal cells early in life, as it is associated with gut closure. Dams were dried off approximately 45 d before expected calving and randomly assigned to 1 of 2 treatments: heat stress (HT) or cooling (CL). During the dry period all cows were housed under shade in a freestall barn, where the pen for CL cows was equipped with active cooling, including water soakers and fans, whereas the pen for HT cows had no soakers or fans. Using rectal temperature and respiration rate as indicators, heat stress was severe. Average rectal temperature in HT cows was 39.3°C compared with 39.0°C in CL cows, and HT cows had a respiration rate of 66.7 breaths/min compared with 43.2 breaths/min for CL cows. Bull calves (n = 30) were immediately separated from their dams at birth, weighed, and then killed before colostrum feeding  or at 1 or 2 d of age following colostrum feeding (n = 5/treatment per day). After slaughter, the small intestine was removed and weighed, and samples from the jejunum were fixed for immunohistochemistry. Birth weight of bulls from HT dams was 1.1 kg lower than that of bulls from CL dams. Thymus, spleen, and heart weights of HT bulls were lower relative to those of CL bulls, whereas liver weight of HT bulls tended to be lower relative to that of CL bulls. Jejunal cell apoptosis decreased with age in both HT and CL calves after birth, mirroring gut closure. However, in utero heat stress increased the apoptotic rate in the jejunum, particularly at birth. We conclude that the chronic exposure to heat strain of HT compared with CL dams in late gestation significantly affected fetal growth and immune tissue development, which may be associated with reduced immune function in early life. Also, late-gestation heat stress increased calves' intestinal apoptosis in the first 2 d of life, which might explain the decreased IgG uptake and limited passive immune competence observed in previous studies. AEA) and cell-mediated immune function, compared with calves born to cows that were cooled when dry  cows was equipped with active cooling, including water soakers over the manger and fans over the manger and stalls, whereas the pen for heat-stressed (HT) cows had no soakers or fans. The average temperature-humidity index of the pens housing the pregnant cows was 77.9 and 77.6 for CL and HT, respectively, in 2014 and 77.6 and 77.9 for CL and HT, respectively, in 2015. Rectal temperature was measured using a digital thermometer every day at 1400 h . Respiration rate was determined by visual observations of flank movements for 1 min, and measurements were made every day at 1400 h. Feed and water were available ad libitum for all cows at all times. All calves were raised postnatally under identical management in stalls under shade.Animal use was approved by the University of Florida Institutional Animal Care and Use Committee. The experiment was conducted during the summer over 2 consecutive years (2014 and 2015) at the University of Florida Dairy Unit . All treatments of cows and the resultant calves were assigned randomly and balanced across both years. During the dry period, all cows were housed under shade in a freestall barn, where the pen for cooled (Cows were cooled (n = 40) or heat stressed (n = 40) during an approximately 45-d dry period that represented approximately the final 45 d of pregnancy. After birth, all bull calves were immediately separated from their dams and weighed. Bull calves (n = 30) were killed using a captive bolt to stun the animal followed by exsanguination at birth without colostrum feeding (n = 5/treatment) and at 1 and 2 d of age following colostrum feeding (n = 5/treatment per day). To avoid confounding effects of maternal colostrum, pooled good-quality colostrum  was fed by esophageal tube according to University of Florida Dairy Unit standard operating procedures within 4 h after birth to bulls slaughtered on 1 and 2 d of age. Calves killed on 1 and 2 d of age received 4 L of pasteurized whole milk daily. After slaughter, the wet weight of the small intestine and other organs, including the thymus, spleen, pancreas, liver, heart, kidney, and lung, was recorded. The small intestine was removed and washed with PBS, and a tissue sample from the jejunum was collected for immunohistochemistry.l-lysine. To investigate the relationship of enterocyte apoptosis and compromised passive immunity after late-gestation heat stress, terminal deoxynucleotidyl transferase dUTP nick end labeling was performed on sectioned jejunal samples to estimate the apoptotic rate of intestinal cells using the same procedure described in 2O2 in PBS for 10 min. Following incubation in equilibration buffer  for 10 min, sections were incubated with terminal deoxynucleotidyl transferase (333 U/mL) and digoxigenin-conjugated nucleotides (Millipore) for 60 min at 37°C. Reactions were stopped by stop/wash buffer and then washing extensively with PBS. Labeled DNA was detected with anti-digoxigenin peroxidase  for 30 min at room temperature followed by colorimetric detection with 3,3′-diaminobenzidine. Sections were counterstained with methyl green, dehydrated, and mounted, and counting was done under a 40× objective on a Nikon Optiphot microscope (Nikon Corp.). Apoptosis was estimated by counting the number of positive cells in a field  to calculate the percentage of positive cells out of total cells in 6 different sections per slide per calf.Jejunal samples for immunohistochemical analysis were fixed in 4% neutral formalin overnight at 4°C and then transferred to 70% ethanol until embedding. Samples were dehydrated and embedded in paraffin  following standard protocols and sectioned at 4 µm onto slides coated with poly-P < 0.05, whereas tendencies were declared at P < 0.15. Heat stress during the dry period increased cow rectal temperature  as well as respiration rate relative to cooling . Therefore, as expected, heat stress during the dry period decreased bull birth weight . Cow or calf was considered a random variable, and treatment, time, and treatment × time were considered fixed effects. In addition, if there was a treatment × time interaction, the pdiff mean separation test was used to compare HT and CL at each individual time. Data are presented as least squares means ± standard error of the mean. Significance was declared at P = 0.02 and P < 0.01, respectively) as well as the heart (P = 0.03). Additionally, in utero heat stress tended to decrease liver weight (P = 0.09) compared with in utero CL calves . Likewise, there was a slight tendency for a treatment × day effect on liver and kidney weights  of the dams that did not have access to fans and soakers indicate that heat stress was severe in the current study. Therefore, as expected, we observed a difference between treatments in the birth weight of calves, with HT calves having lower birth weight relative to CL calves. This observation is consistent with previous studies and likely reflects reduced placental function and reduced gestation length in the cows exposed to heat stress in late gestation . Indeed,Not only were birth weights reduced by in utero heat stress, but individual organ mass was also lower than that in CL calves. In utero heat stress reduced heart weight and tended to decrease liver weight compared with CL calves. Likewise, there was a tendency for treatment × day interaction on liver and kidney weights, whereby in utero HT calves had smaller liver and kidney mass relative to in utero CL calves, particularly at 2 d of age. As heat stress of the dam in late gestation compromises placental and fetal development, fetal heart, liver, and kidney weights are reduced, which is consistent with observations in sheep. For example, Calves, like all young animals, are prone to elevated rates of mortality and morbidity in the neonatal period, and preterm birth may further exacerbate that problem . Early-lConsistent with the overall effect of in utero heat stress on BW, there was an effect of in utero heat stress on immune organ weight, such that in utero HT calves had lower thymus and spleen weights compared with in utero CL calves. This decrease in immune organ weight may result in compromised postnatal immune function, as significant functional outputs of the adaptive immune system are generated in bone marrow and the thymus . In addiFollowing colostrum ingestion, macromolecules such as immunoglobulins are absorbed at the intestine through a neonatal Fc receptor-independent and nonselective process , and theColostrum feeding may also affect the timing of gut closure. In conclusion, the substantial difference in heat strain on HT versus CL cows during late gestation significantly affected overall growth in utero, fetal primary lymphoid tissue development, and intestinal apoptosis. Late-gestation heat stress increases jejunal apoptosis in the first 2 d of life, indicating that in utero heat stress may accelerate gut closure relative to CL and is associated with decreased IgG uptake and limited passive immune competence. Consequently, decreased IgG uptake and reduced immune function following gestational heat stress may lead to reduced health and growth of the calf in the long term. Of particular concern from a management perspective is the observation that this acceleration of gut closure appears to occur even before birth and before colostrum consumption. Thus, it may be challenging to reverse after parturition, so management efforts should focus on cooling the dam during late gestation.


Setting a proper SCC cut-off value for determining the intramammary infection status of each individual cow is essential to early mastitis detection and prevention. In this study, we compared the effect of two commonly used SCC thresholds on distinguishing milk microbiota and host gene expression patterns, and demonstrated that the microbial composition and peripheral blood leukocyte transcriptome profiles did have conspicuous differences between cows with SCC above and below 100,000 cells/mL, respectively, which may help establish why 100,000 cells/mL is a more suitable cow-level cut-off value of subclinical mastitis diagnosis than 200,000 cells/mL from the perspective of microbiota and transcriptomic responses. Bifidobacterium and Lachnospiraceae_AC2044_group, which were more abundant in milk samples from cows with SCC below 100,000 cells/mL. As for the transcriptome profiling, 79 differentially expressed genes (DEGs) were found to have the same direction of regulation in two sites, and functional analyses also showed that biological processes involved in inflammatory responses were more active in MSCC and HSCC cows. Overall, these results showed a similarity between the milk microbiota and gene expression profiles of MSCC and HSCC cows, which presented further evidence that 100,000 cells/ml is a more optimal cut-off value than 200,000 cells/mL for intramammary infection detection at the cow level.Somatic cell count (SCC) is an important indicator of the health state of bovine udders. However, the exact cut-off value used for differentiating the cows with healthy quarters from the cows with subclinical mastitis remains controversial. Here, we collected composite milk (milk from four udder quarters) and peripheral blood samples from individual cows in two different dairy farms and used 16S rRNA gene sequencing combined with RNA-seq to explore the differences in the milk microbial composition and transcriptome of cows with three different SCC levels . Results showed that the milk microbial profiles and gene expression profiles of samples derived from cows in the MSCC group were indeed relatively easily discriminated from those from cows in the LSCC group. Discriminative analysis also uncovered some differentially abundant microbiota at the genus level, such as  Subclinical mastitis is known as one of the most prevalent mammary diseases plaguing the dairy industry. It can have adverse effects on the milk quality and cause economic losses. Early diagnosis of bovine subclinical mastitis is difficult, as there are no clinical signs. Fortunately, the association between abnormal somatic cell counts and the incidence of clinical or subclinical mastitis has been identified and for now, somatic cell count monitoring is still used as an effective method for subclinical mastitis detection ,2. ThereKlebsiella and Streptococcus higher in cows suffering from subclinical mastitis [Cutibacterium, Colidextribacter, and Paeniclostridium has also been shown to be correlated with adverse udder health [To study the microbial composition shift caused by diseases and to help us understand the roles of the microbiota in the development of some bacteria-caused diseases, 16S rRNA gene sequencing was intensively utilized. Compared with traditionally used quantitative PCR (qPCR) for mastitis pathogen detection, 16S rRNA can almost characterize the entire bacterial profile, which is more conducive to studying subclinical mastitis, because research has indicated that commensal and potential beneficial bacteria are also involved in the subclinical mastitis incidence rate . By usinmastitis . Apart fr health , suggestGlobal functional genomics technologies enable us to explore the dynamic changes in response to the environmental stimulus and underlying diseases. RNA-seq, which can simultaneously quantify the expression of a large number of genes, is quite suited for investigation of the complex host–pathogen interplay occurring during mastitis . SignifiAs indicated, both milk microbiota and the host transcriptional profile can help investigations of the differences between healthy and infected cows. Therefore, the objectives of this study were to utilize RNA-seq and 16S rRNA gene sequencing to determine whether using 100,000 cells/mL as an SCC cut-off value for intramammary infection diagnosis is better than using 200,000 cells/mL at the cow level and explore the underlying reason from the perspective of the bacterial community and global gene expression. In this study, sample collection was conducted in two well-managed commercial dairy farms located in two different cities  in China and in two different seasons. To minimize the effect of recent mammary infections, all cows (Holstein cows) selected in this study were free of clinical mastitis and antibiotic treatment for at least 2 consecutive months before the sample collection. Milk samples were collected aseptically, following cleaning of teat ends with 70% ethanol. Milk from each of the 4 quarters was evenly mixed in a 100 mL-sterile tube after discarding the first few streams of milk. Samples were kept on dry ice during the whole collection and transfer process and were later stored at −80 °C until DNA extraction. A total of 121 milk samples were divided into the following three groups based on SCC value: low SCC group (LSCC): <100,000 cells/mL; medium SCC group (MSCC): 100,000–200,000 cells/mL; high SCC group (HSCC): > 200,000 cells/mL. A blood sample was aseptically taken from the caudal vein of each cow and stored in a 10-mL pre-added EDTA-K2 vacutainer on the same day the milk samples were collected.® DNA PCR-Free Sample Preparation Kit  following the manufacturer’s recommendations. Amplification was performed under the following conditions: denaturation at 98 °C for 1 min, then 30 cycles of denaturation at 98 °C for 10 s, followed by annealing at 50 °C for 30 s, elongation at 72 °C for 30 s and a final step at 72 °C for 5 min. Library quality was assessed on a Qubit@ 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system. Qualified library was later sequenced on Illumina NovaSeq 6000 platform and generated 250 bp paired-end reads.Total genome DNA was extracted from milk using the cetyltrimethylammonium bromide (CTAB) method. The purity of DNA was assessed by 1% agarose gel electrophoresis. The V3-V4 hypervariable region of bacterial 16S rRNA genes was then amplified using universal primers 341F (5′-3′: CCTAYGGGRBGCASCAG) and 806R (5′-3′: GGACTACNNGGGTATCTAAT) with the barcode to tag the PCR products from each sample. PCR products were mixed in equidensity ratios to generate a mixture which was then purified with the Qiagen Gel Extraction Kit . The purity and integrity of the PCR products were evaluated using Agilent5400 . Sequencing libraries were generated using the TruSeqMicrobiota profiling was carried out following Qiime2 (v 2020.8) pipeline. Amplicon sequences were assigned to each sample based on the tagged barcode. Quality control was accomplished by cutting off the adaptors and using Divisive Amplicon Denoising Algorithm (DADA2) to correct sequence errors, filter chimeric sequences, and merge the paired-end reads with a minimum overlap of 30 nt. High-quality reads that survived from the filtration were summarized to generate an amplicon sequence variant (ASV) table. A Naïve Bayes classifier (a plugin in Qiime2) was trained based on sequences extracted from the SILVA database release 138 and was then applied to ASV to perform taxonomic classification. Contaminating mitochondrial and chloroplast sequences were removed from the feature table. Downstream microbiota analyses were conducted using the R package vegan. Rarefaction curves of richness were generated at the genus level to test the adequacy of the sequencing depth. A total of 5 α indices  were calculated to estimate alpha diversity. Pairwise comparisons of the alpha diversity indices were conducted by constructing a linear mixed model to correct site effect, lactation stage, and parity using the R package lmerTest (v 3.1.3) and emmeans. The parity and lactation stage were classified into two levels  and late (>100 d) for lactation stage) as research suggested that parity and stage of lactation may have an effect on overall diversity of the udder microbiota . The sitp < 0.05. Additionally, Gene Set Variation Analysis (GSVA) was conducted to confirm the result of enrichment analysis and further estimate changes in the activity of biological process (BP)-related pathways.Total RNA was isolated from peripheral white blood cells using Trizol reagent  following the manufacturer’s instructions. Quality and integrity check were performed using Nanodrop  and Aglient 2100 BioAnalyzer , respectively. Sequencing was conducted on the Illumina NovaSeq 6000 platform  and generated 150 bp paired-end reads. Adapters and reads with low quality and a length less than 36bp were removed by Trimmomatic (v. 0.39) before the alignment to the bovine reference genome ARS-UCD1.2 using STAR (v. 2.7.9a). A total of 36 transcriptomic datasets were included in this study (18 per site and 6 per group). Reads mapped to the reference genome were quantified by feature Counts in subread (v. 2.0.2) package. Genes with low expression levels (counts per million < 1) were removed from the downstream analysis. Differentially expressed gene (DEG) analysis was performed separately for samples collected from different sites using R package DESeq2 with the covariates mentioned before being corrected. Read count normalization was accomplished using rlog transformation. Functional analyses including KEGG and Gene Ontology (GO) enrichment analysis were carried out using the R package Clusterprofiler (v 3.18.1). To merge the transcriptomic data from the two sites to conduct the following analysis, the batch effect was removed using the R package limma (v 3.46.0). To test for the associations between differentially abundant taxa and top differentially expressed genes, the R package MaAsLin was used to assess the relationship between DEG and microbiota with the following model: microbial abundance~top DEGs + site + parity + lactation stage. Significance was determined at The rarefaction curves of all samples reached a plateau, suggesting a sufficient amount of sequencing data . Four saFirmicutes (0.42 ± 0.14), Bacteroidota (0.21 ± 0.07), Actinobacteriota (0.20 ± 0.08), Proteobacteria (0.08 ± 0.08), and Patescibacteria (0.05 ± 0.05) and the top 10 most abundant genera were Oscillospiraceae_UCG-005, Corynebacterium, Bacteroides, Ornithinimicrobium, Christensenellaceae_R-7_group, Oscillospirales_UCG-010, Paracoccus, Romboutsia, Rikenellaceae_RC9_gut_group, and TM7a, constituting more than 33% of the total relative abundance  significantly higher than those from site1 , suggesting the core phyla of the milk microbiota may vary from site to site.Taxonomic annotation identified 34 phyla and 755 genera in total from the 87 composite milk samples. As displayed in bundance b. NotablTo evaluate the difference in alpha diversity of milk microbial composition in LSCC, MSCC, and HSCC groups, we chose the linear mixed model with the smallest Akaike information criterion (AIC) to correct covariates that might affect the result. Unfortunately, there was no significant difference in alpha diversity among the three groups using any of the five alpha indices after correction for cofounding factors . The adjp = 0.41). Conversely, significant difference in the composition existed between the HMSCC and LSCC groups (p < 0.02).Between-group composition variation was significant for analysis of milk microbiota by group and was performed using groups LSCC and HSCC and groups LSCC and MSCC. By merging LSCC and MSCC into one single group (LMSCC), the composition between LMSCC and HSCC did not significantly differ . Group factor was selected as a dependent explanatory variable when analysis was conducted using (1) LSCC and MSCC groups; (2) LSCC and HSCC groups; (3) HMSCC and LSCC groups. Together, these results demonstrated that the microbial composition of MSCC shared more similarity with that of HSCC, and using a cut-off value of 100,000 cells/mL could discriminate the variation in microbial composition.A stepwise forward selection procedure was performed for each model that incorporated different pairs of groups. The result further confirmed that site had the greatest effect on milk microbiota composition and explained over 20 percent of variation in all models (Rp < 0.05), among which 27 were observed to be more prevalent in LSCC and 6 in MSCC. For HSCC vs. MSCC comparison, only one differentially abundant genus  was detected and had higher abundance in MSCC. To identify the most relevant bacteria in each group, we performed pairwise comparative analysis between any two groups at the genus level. As before, we also merged LSCC and MSCC or HSCC and MSCC into one group to see which cut-off value could yield more differentially abundant genera. Only bacteria taxa present in at least 20% of the samples used in comparisons were kept for the differential abundance analysis. A total of 33 differentially abundant genera were identified in LSCC vs. MSCC comparison (n = 29) than in LMSCC vs. HSCC comparison (n = 3), indicating that the microbial signature of MSCC cows was more different from that of LSCC than that of HSCC cows (Bifidobacterium and Lachnospiraceae_AC2044_group) (padj < 0.05) that were most significantly differentially abundant in LSCC vs. MSCC comparison were also identified as differentially abundant genera in LSCC vs. HSCC comparison. In addition, the relative abundance of these two genera was higher in the group with lower SCC (p > 0.05). The result also showed that more differentially abundant genera existed in LSCC vs. MSCC comparison than in HSCC vs. MSCC comparison, and in HMSCC vs. LSCC comparison  were shared in all comparisons and more abundant in the group with a lower SCC level (n = 104) than in MSCC vs. HSCC (n = 57), and in HMSCC vs. LSCC comparison (n = 29) than in LMSCC vs. HSCC comparison (n = 3). To investigate the differences in the metagenome function contents among the three groups, we used predicted metabolic pathways with prevalence of no less than 30% for comparison analysis. Using the same method as differentially abundant genera identification, we found that three metabolic pathways  and only kept the top 100 most abundant genera with prevalence > 60%. Nodes with a significant correlation > 0.8 were kept for network visualization . Networkadj2= 0.18, p < 0.001)  .p < 0.05 and |log2(FoldChange)| ≥ 1. In samples from both sites, more DEGs were identified in LSCC vs. MSCC (363 for site1 and 955 for site2) than in HSCC vs. MSCC (63 for site1 and 270 for site2). Furthermore, cluster analysis and expression heatmap of the most significant DEGs (FDR < 0.1) in both sites revealed apparently distinct transcriptional profiles between LSCC and the other two groups   . Unfortup < 0.05) were also mainly involved in the cytokine-mediated signaling pathway and positive regulation of inflammatory response. In addition, GSVA further confirmed immune-related biological processes, such as regulation of interleukin 6 mediated signaling pathway, B cell chemotaxis, and positive regulation of T cell apoptotic process, were lowly expressed in LSCC, compared with the other two groups , suggesting that these genes play an important role in inflammatory response. Significant enriched biological processes in GO terms (o groups a–c.p < 0.05)  a. Among CC level b.Early diagnosis of subclinical mastitis is crucial for therapeutic decisions and for now, SCC is still broadly used as a proxy for subclinical mastitis . To our Though the mammary gland is the organ directly involved in the synthesis and secretion of milk, it is not always the most ideal material to study intramammary diseases due to collection difficulty and effect on the lactating cows. Blood, however, is relatively easy to collect and is responsible for various substance and molecule exchanges with the mammary gland. Moreover, the presence of some immune cells within the blood, such as leukocytes, enables blood transcriptome to reflect the overall physiological condition of cows ,17. TherBifidobacterium and Lachnospiraceae_AC2044_group in milk with SCC < 100,000 cells/mL. Previous studies have referred to Bifidobacterium as one of the markers of “healthy microbiota” and shown that it has lower relative abundance in cows with udder pathology [Bifidobacterium breve was reported to have an effect on clearance of minor mastitis causing pathogens from infected quarters [Lachnospiraceae_AC2044_group has not been identified as a marked beneficial bacterium in uninfected mammary quarters before. However, a higher percentage of unclassified Lachnospiraceae was detected in milk samples derived from healthy quarters in one study [The present study identified an enrichment of genera athology . Additioquarters . Genera For the first time, the topological properties of the co-occurrence networks in milk microbiota were compared among groups with different SCC levels to illustrate how microbial interactions change with the increase in SCC. Closeness centrality represents the tightness of the network, and a higher closeness centrality usually indicates the greater importance of the node . While bSamples from two different farms were used in this study to provide a result that may have a wider application. However, correcting batch effects for microbiota studies remains a problem because the same methods that work well for other types of omics data are not necessarily appropriate for microbiome datasets, which are extremely sensitive to the variation in experimental and computational processing . The ALDS100A9 has previously been shown to be actively involved in inflammatory processes and upregulated in bovine mastitis [Muribaculaceae, a beneficial commensal bacteria demonstrated by several studies, had a lower relative abundance in cows with subclinical mastitis [S100A9 in this study. Thus, we speculated that Muribaculaceae may play a regulatory role in inflammation by interfering with the expression of S100A9. Additionally, we found another gene–bacteria pair that might serve the same purpose. GADD45G was enriched in NF-κ B and the MAPK signaling pathway was highly expressed in HSCC group. Though the genera (Oscillospiraceae_UCG-005) correlated with this gene have not been proven to be directly linked to subclinical mastitis before, their relative abundance did decrease in some other diseases [In this study, the correlation between the top DEGs and differentially abundant genera were estimated to help understand their roles in host–microbe interplay. S100 calcium-binding protein A9 encoding gene mastitis ,27. Furtmastitis ,29 and wdiseases ,31. With regard to the number of DEGs and differentially abundant genera, samples from LSCC were more easily discriminated from samples in MSCC. However, the number of differentially abundant genera in LSCC vs. HSCC comparison was smaller than in LSCC vs. MSCC. We conjectured that this was because some underlying factors may have unequal effects on certain milk microbiota derived from cows with different SCC levels, and the relationship between the abundance of some bacteria and SCC was not always linear . To our knowledge, this is the first study to integrate host transcriptomics and microbiome to explore the cow-level SCC cut-off value for subclinical mastitis diagnosis. The results revealed a visible difference in microbiota profiling and gene expression pattern between composite samples with SCC above and below 100,000 cells/mL. Using 200,000 cells/mL as the threshold, however, led to a negligible difference in the intermediate molecular phenotypes, indicating 100,000 cells/mL can be more discriminative than 200,000 cells/mL. Though considerable effort has been devoted to finding the most reliable SCC threshold to predict mammary gland infection, it should be noted that the variability in SCC could be explained by different explanatory factors, and according to a previous study, sources of variability should be studied within and between animals before drawing a conclusion on a given animal’s health status . As mentIn conclusion, our study pointed out the potential superiority of using 100,000 cells/mL as the cow-level threshold of subclinical mastitis detection over 200,000 cells/mL in terms of the changes in microbiota and transcriptome. Furthermore, the results showed that some beneficial bacteria may be helpful for alleviating subclinical mastitis progression. Thus, the differentially abundant genera and differentially expressed genes found in the current study might be conducive to the future diagnosis of intramammary infection.


Objective: Surveying public awareness of antibiotic use and antibiotics can identify factors relevant to the design of effective educational campaigns. The aim of this study was to evaluate the knowledge, attitudes, and practices related to antibiotic use and multidrug-resistant pathogens in the general population in Pakistan.Research Design and Methods: Cross-sectional survey was conducted, using a 60 itemed structured questionnaire and recruited individuals by convenient sampling from the general population in the four provinces of the country. Descriptive statistics were used to evaluate the responses and the chi squared statistic was used to assess differences between groups.Results: The response rate was 87.6% . Half of the respondents had received at least one prescription of antibiotics in the 6 months preceding the survey. Knowledge about antibiotic use, (39.8%) individuals scored above the mean (≥3) showed good knowledge about antibiotic use. Urban residents and male showed significant higher knowledge (p < 0.001) about antibiotic use. Approximately 50% of the respondents correctly answered the question about antibiotic resistance. Of the 3,611 received antibiotics, 855 (23.7%) were indicated for cough, 497 (13.8%) for a sore throat, 335 (9.3%) for ear ache, 665 (18.4%) for a burning sensation during urination, 667 (18.4%) for wounds or soft tissue inflammation. MDR pathogen was perceived as an important topic by  60.1% of respondents.Conclusion: Participants were aware of the problem of multidrug-resistant pathogens and understood the responsibility of each individual to avoid the spread of these infectious agents. The development of antimicrobial resistance (AMR) is a major public health concern in Pakistan  and worlIn some developing nations, Gram-negative bacteria are a major cause of neonatal sepsis , and theA considerable number of studies have shown that the general population plays a key role in spreading the AMR due to lack of awareness about antibiotics among population in Pakistan . The WorWe sampled all four provinces of Pakistan . The provinces consist of divisions, districts, tehsils (administrative areas containing towns), and villages. Three districts were randomly selected from Punjab and Sindh and two each from Khyber Pakhtunkhwa and Baluchistan. From each target district, we selected one city and one village. City was conveniently selected by the availability of data collectors. We performed a cross-sectional survey in the four provinces of Pakistan. We used convenient sampling and try to collect data from maximum participants during May to November, 2020. Total 7,631 participants approached in the study during this time frame. Participants who were Immigrants, individuals over 80 or under 18 years, and persons with cognitive impairment (N = 479), participants initially participated in the study and during study refused to continue the study due to personal issues  (N = 219), and missing data from survey (N = 379) were excluded from the study. Finally, sample of 6,684 participants was included in the study. . IndividSixteen data collectors were trained to conduct the research in the provinces. Most data collectors were pharmacy students in their final year of study, who were supervised by teachers from the local university. Training covered the following aspects: 1) presenting a concise introduction of the study rationale to respondents; 2) conducting face-to-face interviews; 3) coping with difficulties in data collection, such as lack of cooperation. The training lasted 3 days and included a demonstration by the primary researcher.The knowledge, attitudes, and practices regarding antibiotic use covered four topics: 1) exposure to antibiotics, evaluated by two questions about symptoms and prescription in the preceding 6 months. 2) Knowledge about antibiotics, evaluated by responses to seven statements related to correct identification of antibiotics and to opinions regarding antibiotic use in general. 3) Attitudes and practices related to antibiotic use, assessed by seven items concerning how participants generally obtained antibiotics and whether they were concerned about antibiotic resistance. 4) Whether participants requested antibiotics for personal use, how they used antibiotics, and whether they developed side effects during use (six items). The questionnaire used in this study differed in its contents from those of previous studies . The secCase 1: Let us suppose that your neighbor, an elderly person residing alone, requires some help and you have been shopping for him for a couple of months. After a hospital stay, he tells you that he has become infected with a hospital-acquired pathogen. How would you behave with him/her?Case 2: Let us suppose that your coworker, with whom you share an office and some office items, tells you after a hospital stay that she has become infected with a hospital-acquired pathogen. How would you behave with him/her?Each case scenario was associated with eight statements, and response options were “strongly agree,” “agree,” “somewhat disagree,” and “disagree.”In addition, demographic information  was also collected and included in the analysis. The questionnaire was modified slightly from a previous study  accordinp < 0.05 was considered significant.Descriptive analysis was performed for the demographic variables. To analyze the knowledge about antibiotics, each correct response was awarded one point. For a positive question, the responses “strongly agree” or “agree” were calculated as one point and “do not know,” “disagree,” or “strongly disagree” were calculated as zero. For a negative question, “disagree” or “strongly disagree” were calculated as one point and “strongly agree” or “agree” and don’t know were calculated as zero. A cumulative score of the seven knowledge items on antibiotics and the four items on MDR was derived in the same manner. The cut point of ≥3 is considered to be good knowledge for the knowledge about antibiotic as it was also chosen in the previous studies that those who scored more than mean were considered in good range. A complete questionnaire is subjected to the analysis those with the missing data were excluded in screening phase. Percentages for each item were calculated and the chi squared statistic was used to check significant differences between demographic groups. The research was carried out in accordance with the tenets of the Declaration of Helsinki and was approved by the Medical Research Ethics Committee of Xi’an Jiaotong University, Shaanxi, China, approved reference number (XJTMD11-2020). Respondents who were literate provided a signed consent form and for those who were not literate, the data collector signed the form on their behalf after explaining the content of the form.Out of 7,631 individuals who were approached for the study, 6,684 (87.6%) completed questionnaire items on antibiotic use and MDR pathogens. The mean age (± standard deviation) of the respondents was 32.97 (10.8) years, 4,746 (71.0%) were male, 772 (11.5%) had education above post-graduation and 2,833 (42.4%) resided in rural areas .p < 0.001). Approximately half of the respondents correctly answered the question about antibiotic resistance. The mean (± standard deviation) of the knowledge score of antibiotic use was 2.51 (1.80) and 2,260 (39.8%) individuals scored above the mean (≥3). Few participants provided correct answers to the questions about antibiotics, and those tended to be urban residents and male showed significant higher knowledge (p < 0.001). Of the 3,611 who received antibiotics, 855 (23.7%) were indicated for cough, 497 (13.8%) for a sore throat, 335 (9.3%) for ear ache, 665 (18.4%) for a burning sensation during urination, 667 (18.4%) for wounds or soft tissue inflammation, and 677 (18.5%) for other reasons. Almost two thirds  of those who were prescribed antibiotics stopped the therapy before completion, because 433 (18.6%) felt better, 544 (23.3%) were worried about side effects, 78 (3.3%) experienced a side effect, 455 (19.5%) forgot to take the medication, 211 (9.0%) experienced “too much stress,” and 611 (26.2%) for other reasons. Almost two thirds 4,211 (63%) of all participants had heard of MDR pathogens, 7.3% 488) reported that they knew an individual who had tested positive for an MDR pathogen, and 12% (802) reported that they themselves had tested positive for an MDR pathogen once in their lifetime. Fifty-four percent of respondents  reported that they were prescribed antibiotics within the preceding 6 months, with a significantly higher proportion of participants from Sindh  than Khyber Pakhtunkhwa  reported sharing medications with family members and 35.8% 2,393) reported that they had antibiotics at home to use when necessary. MDR pathogen was perceived as an important topic by  60.1% of respondents . In termThe first scenario involved an aged neighbor hospitalized with an MDR pathogen infection and the second scenario involved a coworker testing positive for an MDR pathogen. For the first scenario, half of the respondents demonstrated good hygiene practices: 50.6% would wash their hands, 51.8% would disinfect their hands, 49.3% would change clothes after interacting with the neighbor, and 41.8% would avoid the neighbor completely . For theIn this study, we assessed the knowledge and opinions of the general public in Pakistan about antibiotic use and MDR pathogens by interviewing the participants. Individuals showed poor knowledge of antibiotic use and poor medication adherence. Similarly, knowledge about MDR pathogens was scant, but fear of the effects of MDR pathogens was more pronounced.More than half had been prescribed antibiotics in the past 6 months, a lower percentage reported for the United Kingdom (38%) and Germany . The maiStaphylococcus aureus in Scotland  and in Scotland (86%) . Very feScotland .We did not find a significant difference in antibiotic use scores between urban and rural residents, in contrast with the previous study in Pakistan  or with More than half of the participants reported that they were personally concerned about infection with a MDR pathogen, but the answers to the case scenarios illustrated that the majority of individuals exhibited reasonable and non-stigmatizing behavior toward carriers of MDR pathogens in their vicinity. This finding is remarkable, considering that more than half of the participants had stated that they were fearful of contracting MDR pathogens. The percentage of respondents that reported they would avoid the infected person was higher than that reported by Raupach-Rosin and colleagues . A largeThe strength of our study is the comparably large sample and the fact that the study population was sampled from the general public. However, because the knowledge of antibiotics and MDR pathogens was assessed by closed-ended questions, respondents may have selected the most favorable answers rather than accurate ones, and therefore, a qualitative approach might be more suitable to reveal misconceptions. Our study faced some limitations as well such as we selected the districts randomly and targeted the cities, village and participants from the city conveniently. Also, that participants may have given socially desirable answers, rather than actual practices.General knowledge about antibiotic use and MDR pathogens was poor in the population we sampled. However, there was a high awareness about antibiotic resistance as a public health problem, although only a few respondents reported to have been personally affected by MDR. Therefore, information about the implications of MDR pathogens should be made available for the general public. Information campaigns about correct antibiotic use could increase knowledge, thereby improving appropriate practices in antibiotic use and resistance in both humans and livestock. The data in this study provide new information and may serve as the basis for development of interventional programs and policies.We recommend strict government policies should be implemented at hospital level and community pharmacy level to prescribe and dispense antibiotics in the presence of qualified physician and pharmacist. Standard guidelines and proper trainings should be provided to physicians and other health care providers to promote the safe prescribing of antibiotics to avoid the antimicrobial resistance. Community based interventions and programs should be initiated to increase the awareness about antibiotic resistant among general population. Government should increase the awareness and initiate the campaigns on electronic media for safe use of antibiotic in general population. Collective efforts of government, stakeholders, health care providers, patients, researchers, pharmaceuticals companies, policy makers and drug regulatory bodies will help to combat the life threatening scenario of antimicrobial resistant around the globe.