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Antagonistic interactions among different functional guilds of nematodes have been recognized for quite some time, but the underlying explanatory mechanisms are unclear. We investigated responses of tomato (Solanum lycopersicum) to two functional guilds of nematodes - plant parasite (Meloidogyne javanica) and entomopathogens (Heterorhabditis bacteriophora, Steinernema feltiae belowground, and S. carpocapsae) - as well as a leaf mining insect (Tuta absoluta) aboveground. Our results indicate that entomopathogenic nematodes (EPNs): 1) reduced root knot nematode (RKN) infestation belowground, 2) reduced herbivore (T. absoluta) host preference and performance aboveground, and 3) induced overlapping plant defense responses by rapidly activating polyphenol oxidase (PPO) and guaiacol peroxidase (GP) activity in roots, but simultaneously suppressing this activity in aboveground tissues. Concurrently, we investigated potential plant signaling mechanisms underlying these interactions using transcriptome analyses. We found that both entomopathogens and plant parasites triggered immune responses in plant roots with shared gene expression. Secondary metabolite transcripts induced in response to the two nematode functional guilds were generally overlapping and showed an analogous profile of regulation. Likewise, we show that EPNs modulate plant defense against RKN invasion, in part, by suppressing active expression of antioxidant enzymes. Inoculations of roots with EPN triggered an immune response in tomato via up-regulated phenylpropanoid metabolism and synthesis of protease inhibitors (PIs) in plant tissues, which may explain decreased egg laying and developmental performance exhibited by herbivores on EPN-inoculated plants. Furthermore, changes induced in the volatile organic compound (VOC)-related transcriptome indicated that M. javanica and/or S. carpocapsae inoculation of plants triggered both direct and indirect defenses. Our results support the hypothesis that plants 'mistake' subterranean EPNs for parasites, and these otherwise beneficial worms activate a battery of plant defenses associated with systemic acquired resistance (SAR) and/or induced systemic resistance (ISR) with concomitant antagonistic effects on temporally co-occurring subterranean plant pathogenic nematodes and terrestrial herbivores.

Clostridium perfringens-induced necrotic enteritis (NE) is an economically important disease of broiler chickens. The present study evaluated the effect of C. perfringens on the intestinal histomorphometry, enteric microbial colonization, and host immune responses using 3 experimental NE reproduction methods. The experimental groups consisted of 1) unchallenged Control diet (corn-soybean meal), 2) Control diet + Eimera inoculation at d 11 followed by C. perfringens challenge at d 15 (ECp), 3) Wheat-based diet + C. perfringens challenge (WCp), and 4) Wheat-based diet + Eimeria inoculation followed by C. perfringens challenge (WECp). The results showed that chickens receiving ECp and WECp had reduced (P < 0.05) bird performance coupled with enteric gross lesions and epithelial damage at d 17 and 24 of age compared to unchallenged control birds. These ECp and WECp administered birds also had increased (P < 0.05) ileal colonization by clostridia and E. coli at d 17 and 24, while the resident Lactobacillus counts were reduced (P < 0.05) at d 24 of age. Furthermore, at d 24, jejunal transcription of IL-6, IL-10, annexin-A1 and IL-2 genes was upregulated (P < 0.05) in the ECp group, whereas the transcription of TNF receptor associated factor (TRAF)-3 gene was increased (P < 0.05) in WECp treated birds when compared to unchallenged control group. Additionally, stimulation of chicken splenocytes and cecal tonsilocytes with virulent C. perfringens bacilli or their secretory proteins resulted in a higher (P < 0.05) frequency of T cells and their upregulation of MHC-II molecule, as determined by flow cytometry. These findings suggest that C. perfringens, while inducing epithelial damage and changes in microbiota, can also trigger host immune responses. Furthermore, NE reproduction methods using coccidia with or without the wheat-based dietary predisposition seem to facilitate an optimal NE reproduction in broiler chickens and thus, may provide better avenues for future C. perfringens research.

Background:Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens.Objective:n this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated.Method:Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay.Results:The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH.Conclusion:Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

1. The objective of the study was to investigate the influence of maternal and parent of origin effects (POE) on genetic variation of Iranian native fowl on economic traits. 2. Studied traits were body weights at birth (BW0), at eight (BW8) and 12 weeks of age (BW12), age (ASM) and weight at sexual maturity (WSM), egg number (EN) and average egg weight (AEW). 3. Several models, including additive, maternal additive genetics, permanent environmental effects and POE were compared using Wombat software. Bayesian Information Criterion (BIC) was used to identify the best model for each trait. The chance of reranking of birds between models was investigated using Spearman correlation and Wilcoxon rank test. 4. Based on the best model, direct heritability estimates for BW0, BW8, BW12, ASM, WSM, EN and AEW traits were 0.05, 0.21, 0.23, 0.30, 0.39, 0.22 and 0.38, respectively. Proportion of variance due to paternal POE for BW8 was 4% and proportion of variance due to maternal POE for BW12 was 5%. 5. Estimated maternal heritability for BW0 was 0.30 and for BW8 and BW12 were 0.00 and 0.01, respectively, which shows that maternal heritability was reduced by age. 6. Based on the results, considering POE for BW8 and BW12 and maternal genetic effects for BW0 improved the accuracy of estimations and avoid reranking of birds for these traits.

Cationic antimicrobial peptides are being developed as a promising class of antimicrobial sub-stances. The introduction of a new antibiotic component requires a comprehensive study of its properties so that it can be relied upon to continue laboratory procedures and clinical trials on laboratory animals or human volunteers. Antimicrobial activity of buforin I was evaluated against 15 of the most important pathogenic bacterial and fungal strains. This was followed by assessing anti-biofilm activity, time-dependent inhibitory, thermal stability, plas-ma stability, hemolysis, and cytotoxic activities. The range of obtained MICs was between 4 and 16 μg/mL. The most resistant and most sensitive microbial strains were S. salivarius and C. perfringens, respectively. Buforin I not only inhibited biofilm formation, but also showed a high biofilm radiation activity. Buforin I was stable in human plasma and also at different temperatures including 40, 60, and 80 °C. Although no significant anti-cancer properties were observed for buforin I, the lack of cytotoxicity as well as the lack of hemolytic activity confirm its safety. The high therapeutic index indicated that buforin I has a considerable pharmaceutical potential and can be a reasonable candidate to replace antibiotics or administered in combination with antibiotics to increase the effectiveness as well as reduce the dose of antibiotics.

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal’s pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD–thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 μg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 μg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD–thanatin interaction.

Three hundred and sixty 1-day-old male broiler chicks were randomly allocated to 4 treatments of 6 replicates to evaluate the effects of cLFchimera, a recombinant antimicrobial peptide (AMP), on gut health attributes of broiler chickens under necrotic enteritis (NE) challenge. Treatments were as follows: (T1) unchallenged group fed with corn-soybean meal (CSM) without NE challenge and additives (NC); (T2) group fed with CSM and challenged with NE without any additives (PC); (T3) PC group supplemented with 20 mg cLFchimera/kg diet (AMP); (T4) PC group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). Birds were sampled for villi morphology, ileal microbiota, and jejunal gene expression of cytokines, tight junctions proteins, and mucin. Results showed that AMP ameliorated NE-related intestinal lesions, reduced mortality, and rehabilitated jejunal villi morphology in NE challenged birds. While the antibiotic non-selectively reduced the count of bacteria, AMP restored microflora balance in the ileum of challenged birds. cLFchimera regulated the expression of cytokines, junctional proteins, and mucin transcripts in the jejunum of NE challenged birds. In conclusion, cLFchimera can be a reliable candidate to substitute growth promoter antibiotics, while more research is required to unveil the exact mode of action of this synthetic peptide.

Imprinted genes display biased expression of paternal and maternal alleles in mammals. They are marked through epigenetic process during gametogenesis. Characterization of imprinted genes has expanded our understanding of the regulation and function of genes. In the current study, 22 experimentally validated imprinted genes in bovine (Bos Taurus) were analysed. Several supervised machine learning algorithms and attribute weighting methods were used to find characteristics of different types of imprinted genes and suggest a classification method for finding maternally and paternally expressed genes in bovine. For assessing the best model and comparing attributes in other organisms, we have also conducted a comparative analysis for human and sheep imprinted genes. According to the results of the present study, GC contents 10 and 100 kb upstream, Gly and Gln amino acids, Ile/ATC codon usage, LINE and SINE in 100kbup and length of first intron were significantly different between the maternal and paternal genes in cattle. Considering all species together, we found that GC content 100 kb up, LINE 100 kb up and the frequency of amino acids like Gly, Gln and Met were the most important attributes for identifying the paternal and maternal imprinted genes. These findings could imply conservation pattern in the attributes among these species.

Probiotics are beneficial microorganisms and have long been used in food production as well as health promotion products. Bioengineered probiotics are used to express and transfer native or recombinant molecules to the mucosal surface of the digestive tract to improve feed efficiency and promote health. Lactococcus lactis is a potential probiotic candidate to produce useful biological proteins. The aim of this investigation was to develop a recombinant Lactococcus lactis with the potential of producing phytase. To enhance the efficiency of expression and secretion of recombinant phytase, usp45 signal peptide was added to the expression vector containing phytase gene (appA2) derived from Escherichia coli. Sequencing of recombinant plasmid containing appA2 showed the correct construction of plasmid. Total length of the phytase insert was 1.25 kbp. A Blast search of the cloned fragment showed 99% similarity to the reported E. coli phytase sequence in the GenBank (accession number: AM946981.2). A plasmid containing usp45 and appA2 electrotransferred into Lactococcus lactis. Zymogram with polyacrylamide gel revealed that the protein extract from the supernatant and the cell pellet of recombinant bacteria had phytase activity. Enzyme activity of 4 U/ml was obtained in cell extracts, and supernatant maximal phytase activity was 19 U/ml. The recombinant L. lactis was supplemented in broiler chicken feed and showed the increase of apparent digestibility on phytate phosphorus in the digestive tract and it was same as performance of E. coli commercial phytase.

This study investigated the effects of an antimicrobial peptide (AMP), cLF36, on growth performance and the histophysiological changes of the intestine in E. coli-challenged broiler chickens. A total number of 360 day old male chicks were randomly assigned to 4 groups of 6 replicates as follows: T1) negative control diet based on corn-soybean meal without E. coli challenge and additives; T2) positive control diet based on corn-soybean meal and challenged with E. coli without any additives; T3) positive control diet challenged with E. coli and supplemented with 20 mg AMP (cLF36)/kg diet; T4) positive control diet challenged with E. coli and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. Results showed that T3 improved growth performance and the jejunal morphology of E. coli-challenged chickens similar to those of T4. While antibiotic non-selectively decreased the population of ileal bacteria, AMP increased the population of Lactobacillus spp. and decreased harmful bacteria in the ileum of E. coli-challenged chickens. Supplementing E. coli-challenged chickens with AMP improved the gene expression of immune cells and upregulated the expression of tight junction proteins compared to other challenged groups. In conclusion, although cLF36 beneficially affected growth performance and the intestinal morphology of E. coli-challenged chickens similar to those of the antibiotic group, this AMP drastically improved the intestinal microbiome, immune cells, and junctional proteins compared to other E. coli-challenged birds, and can be nominated as an alternative for growth promoter antibiotics.

This study was conducted to evaluate the effects of apple cider vinegar (ACV) administration on non-specific immunity of serum and skin mucus, growth indices, and activity of digestive enzymes (amylase, lipase, and protease) in Carassius auratus. For this purpose, 180 fish (weighing 7.35 ± 0.19 g) were allocated to 4 treatment groups with 3 replications in a completely randomized design. Fish were fed for 105 days using a basal diet supplemented with 0% (control), 1% (T 1), 2% (T 2), and 4% (T 3) ACV (contained 5% acetic acid). Results showed a significant increase in lysozyme activity, ACH50, and total immunoglobulin of skin mucus in fish fed with T2 diet (p

Human T-cell lymphotropic virus (HTLV-1) and bovine leukemia virus (BLV) are oncogenic deltaretroviruses, which are the cause of adult T cell leukemia/lymphoma (ATLL) and enzootic bovine leukosis (EBL), respectively. In this study, to evaluate the virus-host interactions in the manifestation of the associated malignancy, four pooled RNA samples of each host (three RNAs in each sample) were applied to RNA-seq. Differential expression analyses were conducted separately between ATLL and EBL groups, in comparison with the healthy group, to identify functional Gene Ontology (GO) terms and hub genes, using DAVID database and MCODE plugin in Cytoscape software, respectively. A broad range of effective genes, involved in the ATLL and EBL, was up- and downregulated. In the virus side, in both malignancy, Tax was expressed very low, but the HTLV-1-HBZ and BVL-As2 transcripts were highly expressed. Some upregulated hub genes, IL2, TOP2A, MKI67, TP73, MYC, and downregulated FOS gene family (FOS, FOSB, and FOSL2), are similarly activated in both human and bovine hosts, in related cell cycle and growth factors. Taken together, it seems that in preventing the infections and cell transformations, Tax must be targeted as a viral factor, and shared peptide in virological and immunological synapses as host factors. Therefore, in the malignant stages, HBZ and As2 transcripts along with growth factors, particularly IL-2R-γ and T-bet or TOP2A, and MKI67 should be targeted in both hosts. Additional studies at the protein level are necessary to elucidate the more useful targets for the therapy of these life-threatening diseases.

Mastitis is one of the costliest diseases in dairy farms caused by infection of different microorganisms such as Escherichia coli, Streptococcus uberis and Staphylococcus aureus. Promoters are significantly involved in regulating gene expression and shedding light on the mechanisms of transcriptional regulation in physiological and immunological processes of the infections. Exploiting regulatory elements such as transcription factor binding sites (TFBSs modules) on the promoter region could reveal co-regulated genes, which allow screating regulatory models and executing a cross-sectional analysis on several databases. In this study, the promoter regions of 11 genes associated with contagious mastitis including CCL4, CXCL8, STAT3, IKBKB, MAPK14, NFKBIA, NFKB1, TNF, IL18, IL6, and HCK were investigated to predict the activating regulatory modules on promoters and to discover the key related transcription factors. By exploring the promoter regions, 228 genes were discovered comprising the same transcription factors modules. Out of 228 genes, 36 were validated using five microarray datasets. The promoter research of these genes revealed that as many as 7 down-regulated and 12 up-regulated genes are predictable in the network. The genes whose functions were associated with the initial gene list (11 genes), were identified by DAVID queries with TFBSs models implying that the approach provides a clear image of the underlying regulatory mechanism of gene expression profile and offers a novel approach in designing gene networks in cattle.

The insulin-like growth factor (IGF) axis is fundamental for mammalian growth and development. However, no comprehensive reference data on gene expression across tissues and pre- and postnatal developmental stages are available for any given species. Here we provide systematic promoter- and splice variant specific information on expression of IGF system components in embryonic (Day 48), fetal (Day 153), term (Day 277, placenta) and juvenile (Day 365-396) tissues of domestic cow, a major agricultural species and biomedical model. Analysis of spatiotemporal changes in expression of IGF1, IGF2, IGF1R, IGF2R, IGFBP1-8 and IR genes, as well as lncRNAs H19 and AIRN, by qPCR, indicated an overall increase in expression from embryo to fetal stage, and decrease in expression from fetal to juvenile stage. The stronger decrease in expression of lncRNAs (average -16-fold) and ligands (average -12.1-fold) compared to receptors (average -5.7-fold) and binding proteins (average -4.3-fold) is consistent with known functions of IGF peptides and supports important roles of lncRNAs in prenatal development. Pronounced overall reduction in postnatal expression of IGF system components in lung (-12.9-fold) and kidney (-13.2-fold) are signatures of major changes in organ function while more similar hepatic expression levels (-2.2-fold) are evidence of the endocrine rather than autocrine/paracrine role of IGFs in postnatal growth regulation. Despite its rapid growth, placenta displayed a more stable expression pattern than other organs during prenatal development. Quantitative analyses of contributions of promoters P0-P4 to global IGF2 transcript in fetal tissues revealed that P4 accounted for the bulk of transcript in all tissues but skeletal muscle. Demonstration of IGF2 expression in fetal muscle and postnatal liver from a promoter orthologous to mouse and human promoter P0 provides further evidence for an evolutionary and developmental shift from placenta-specific P0-expression in rodents and suggests that some aspects of bovine IGF expression may be closer to human than mouse.

Genomic imprinting results in monoallelic expression of genes in mammals and flowering plants. Understanding the function of imprinted genes improves our knowledge of the regulatory processes in the genome. In this study, we have employed classification and clustering algorithms with attribute weighting to specify the unique attributes of both imprinted (monoallelic) and biallelic expressed genes. We have obtained characteristics of 22 known monoallelically expressed (imprinted) and 8 biallelic expressed genes that have been experimentally validated alongside 208 randomly selected genes in bovine (Bos taurus). Attribute weighting methods and various supervised and unsupervised algorithms in machine learning were applied. Unique characteristics were discovered and used to distinguish mono and biallelic expressed genes from each other in bovine. To obtain the accuracy of classification, 10-fold cross-validation with concerning each combination of attribute weighting (feature selection) and machine learning algorithms, was used. Our approach was able to accurately predict mono and biallelic genes using the genomics and proteomics attributes.

The genotypes for Beta-Lactoglobulin (BLG) and calpastatin (CAST) were determined by Polymerase Chain Reaction (PCR) and restriction enzyme digestion and genotyped for calpain (CAPN) by PCR-SSCP method in an Iranian breed sheep, Kurdi. Blood samples were collected from 100 pure bred Kurdi sheep from Kurdi breeding station located in Shirvan, Mashhad. The extraction of genomic DNA was based on Guanidin Thiocyanate-Silica gel method. After PCR reaction, amplicons were digested with restriction enzymes MspI and RsaI for beta-lactoglobulin and calpastatin genes, respectively. The beta-lactoglobulin locus had 3 genotypes with frequencies of 0.24, 0.54 and 0.22 for AA, AB and BB, respectively; calpastatin genotypes had 2 genotypes with frequencies of 0.76 and 0.24 for MM and MN genotypes, respectively. Calpain genotypes were analyzed with SSCP method, which had 2 genotypes with frequencies of 0.92 and 0.08 for AA and AB, respectively. Heterozygosity value for beta-lactoglobulin locus was 49% and for calpastatin and calpain loci was very low (24 and 8%, respectively). chi2 test confirmed the Hardy-Weinberg equilibrium for three loci in this population. These data provide evidence that Iranian's Kurdi sheep breed have a variability, which opens interesting prospects for future selection programs, especially marker-assistant selection between different genotypes of different locus and milk and cheese characteristics, gain and meat traits and also for preservation strategies.

A total of 540 Japanese quail eggs were assigned to 9 treatments of 4 replicates to investigate the effect of in ovo injection of threonine (THR) on mucin2 (MUC2) mRNA expression and digestive enzyme activity. Treatments were (non-injected) eggs and those in ovo injected with saline (0.05 or 0.1 ml) with or without THR (5 mg/ml) in two sites (in or under the air sac). On hatch day, 0.05 ml in ovo injected (under the air sac: TUAS) hatchlings were divided into three groups based on NRC recommendations for THR, while all 0.1 ml in ovo injected chicks were removed due to low hatchability. The remaining treatments received the NRC recommended diet until day 10 post-hatch. Treatments had no effect on protease and amylase activities, while TUAS increased MUC2 gene expression. In conclusion, the in ovo injection of THR increased MUC2 gene expression but had no effect on enzyme activity.

Parent-of-origin-dependent (epi)genetic factors are important determinants of prenatal development that program adult phenotype. However, data on magnitude and specificity of maternal and paternal genome effects on fetal bone are lacking. We used an outbred bovine model to dissect and quantify effects of parental genomes, fetal sex, and nongenetic maternal effects on the fetal skeleton and analyzed phenotypic and molecular relationships between fetal muscle and bone. Analysis of 51 bone morphometric and weight parameters from 72 fetuses recovered at day 153 gestation (54% term) identified six principal components (PC1-6) that explained 80% of the variation in skeletal parameters. Parental genomes accounted for most of the variation in bone wet weight (PC1, 72.1%), limb ossification (PC2, 99.8%), flat bone size (PC4, 99.7%), and axial skeletal growth (PC5, 96.9%). Limb length showed lesser effects of parental genomes (PC3, 40.8%) and a significant nongenetic maternal effect (gestational weight gain, 29%). Fetal sex affected bone wet weight (PC1, p 0.0001) and limb length (PC3, p 0.05). Partitioning of variation explained by parental genomes revealed strong maternal genome effects on bone wet weight (74.1%, p 0.0001) and axial skeletal growth (93.5%, p 0.001), whereas paternal genome controlled limb ossification (95.1%, p 0.0001). Histomorphometric data revealed strong maternal genome effects on growth plate height (98.6%, p 0.0001) and trabecular thickness (85.5%, p 0.0001) in distal femur. Parental genome effects on fetal bone were mirrored by maternal genome effects on fetal serum 25-hydroxyvitamin D (96.9%, p 0.001) and paternal genome effects on alkaline phosphatase (90.0%, p 0.001) and their correlations with maternally controlled bone wet weight and paternally controlled limb ossification, respectively. Bone wet weight and flat bone size correlated positively with muscle weight (r = 0.84 and 0.77, p 0.0001) and negatively with muscle H19 expression (r = -0.34 and -0.31, p 0.01). Because imprinted maternally expressed H19 regulates growth factors by miRNA interference, this suggests muscle-bone interaction via epigenetic factors.

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene which is responsible for this disease has a recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101 bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation inherited to him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD-carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.