ramin seighalani

Identifying the strains carrying acquired tetracycline resistance genes is one of the important points in evaluating the safety of probiotic strains. The aim of the present study is to comprehensively evaluate tetracycline resistance in lactobacillus spp. isolated from the digestive system of Iranian backyard chickens.

In the present study, the phenotypic sensitivity patterns of 36 lactobacillus isolates belonging to four species L. reuteri, L. salivarius, L. crispatus and L. johnsonii were evaluated by measuring the Minimum Inhibitory Concentration (MIC) to tetracycline. After that, four tetracycline resistance genes tet(L), tet(M), tet(W) and tet(O) were detected in isolates with phenotypic resistance to this antibiotic based on the polymerase chain reaction method.

As a result, four isolates of L. reuteri, three isolates of L.salivarius, two isolates of L. crispatus and four isolates of L. johnsonii among the studied lactobacillus spp. showed phenotypic resistance to tetracycline. After the detection of tetracycline resistance genes in isolates with phenotypic resistance, the presence of tet(W) gene was confirmed in all investigated isolates. The simultaneous presence of tet(M), tet(L) and tet(W) genes was observed in three studied L. salivarius isolates. It was also found that the phenotypic resistance observed in three isolates of L. johnsoniiABRIG7, L. johnsoniiABRIG14 and L. johnsoniiABRIG24 is caused by the simultaneous presence of tet(O) and tet(W) genes.

This study showed that tetracycline resistance among Lactobacilli isolated from digestive system of Iranian backyard Chickens is due to the presence of single or multiple resistance genes.. We also found that tet(W) is the most widespread tetracycline resistance gene among investigated Lactobacillus isolates. Therefore, local poultry breeders should refrain from arbitrarily using tetracycline, and when the digestive system of domestic chickens is the source for probiotic microorganism selection, the evaluation of resistance to tetracycline from the phenotypic and molecular point of view should be taken into consideration in order to select safe strains.

There is much information about the regulation of gene expression in response to various stresses at the transcriptional level. Nevertheless, there is limited information about this process at the post-transcriptional level. The diversity and complexity of miRNA regulation indicates their importance in biological processes. Many miRNA regulatory modules can form a complex miRNA-mRNA regulatory network. Therefore, research on miRNA-mRNA regulatory networks can provide valuable information for understanding complex biological processes. These data are very important to further study the stress tolerance mechanisms in plants, especially in rapeseed. In this research, the selection of miRNAs related to drought and salinity stress was made by reviewing the articles on abiotic stresses. Then the target genes were identified using the sequences of mature miRNAs and psRNATarget online software. A gene list of 225 identified target genes was prepared using the UniProt database. Their functional pathway was identified utilizing the DAVID bioinformatics database and KEGG database according to default parameters. Investigations showed that these target genes were involved in several biological pathways including ribosome, spliceosome, proteasome, purine metabolism, selenocompound metabolism, and sulfur metabolism. In addition, the STRING database was used to check co-expression genes. Our result indicated the existence of 37 co-expression genes among the identified target genes.

In the recent years, many countries have banned or restricted the usage of antibiotics as growth-promoting, due to their undesirable effects on human and animal health. Using probiotics as an alternative to growth-promoting antibiotics play an important role in improving performance, health and increasing the quality of poultry products. Lactobacilli are the most popular probiotic bacteria that have beneficial effects on host’s performance and health. In this study, the effect of two native strains of Lactobacillus ruteri isolated from the gastrointestinal tract of native chickens in north-western and south-western of Iran were investigated on performance, carcass characteristics, blood parameters and immune response of broilers.

In this experiment, 300 Arbor-Acres® Plus+ male broiler chickens were divided in to 5 treatments, 4 replications (15 chickens per replication), in a completely randomized design. The experimental treatments include: 1- Basic diet as a control treatment, 2- Basic diet + Avilamycin antibiotic, 3- Basic diet + commercial probiotic (Bio-Poul®), 4- Basic diet + Lactobacillus reuteri isolate MG547731 (Plr2), and 5- Basic diet + Lactobacillus reuteri isolate MG547727 (Plr3).

Under the condition of this experiment, commercial probiotics, antibiotic and either Lactobacillus reuteri (Plr2) isolates, significantly improved daily weight gain in final period (p<0.05). Native isolates probiotics and commercial probiotics significantly (p<0.05) reduced the relative weight of gizzard and proventriculus, though no significant (p<0.05) differences were recorded for other carcass components in broiler chickens. After the first injection of SRBC, treatments include antibiotic and Lactobacillus reuteri (Plr2) isolate caused a significant increase of IgM, and after the second injection, Lactobacillus reuteri (Plr2) and commercial probiotic treatments, showed a significant (p<0.05) increase in immunoglobulin G and Total immunoglobulin. The antibody titer against Newcastle vaccine did not show a significant difference between treatments and the control group. There was no significant difference between serum glucose, cholesterol, triglyceride, VLDL and LDL between treatments and the control group. No significant difference was obtained in serum HDL level in probiotic treatments, while antibiotic treatment appeared with a significant decrease in HDL.

Addition of native Lactobacillus reuteri isolates to poultry diets can have beneficial effects similar to commercial probiotics, on performance, immune response and blood parameters of broilers.

This study was conducted to evaluate the effect of different extraction solvents, including hydro-alcoholic (H-A), ethanolic (Et-OH), and methanolic (Me-OH), on chemical composition and synergistic antibacterial activity of Frangula alnus extract against Staphylococcus aureus and Escherichia coli. The chemical composition of the F. alnus bark extract was evaluated using FTIR, UV-Vis spectroscopy, and GC/MS analysis. The synergistic antimicrobial effect of the extracts with Ciprofloxacin (CIP) and Erythromycin (ERY) was evaluated using minimum inhibitory concentrations, combined disc, and checkerboard titration method. F. alnus contained alkanes, alkenes, phenols, alcohols, esters, terpenes, fatty acid, tetrazole, halo-alkane, anthraquinone as well aromatic, and plasticizer compounds in the ethanolic, methanolic, and hydro-alcoholic extracts. Total phenolic compounds for ethanolic, hydro-alcoholic and methanolic extracts were recorded 110.92±0.01, 95.27±0.01 and 126.6±0.02 g/L Gallic acid, respectively. Et-OH and H-A extracts in both combined and synergistic forms significantly inhibited bacterial growth. Concerning the antibacterial activity of the extracts, it was assumed that the compounds present in the plant extracts would destroy the bacterial cell membranes and consequently cause leakage of intracellular contents. Therefore these antibiotics in combination with the extracts would be able to affect their target sites (CIP; DNA gyrase and ERY; 50S subunit) more effectively. According to the results, H-A, Me-OH, and Et-OH extract efficiently in the synergistic state with erythromycin and ciprofloxacin can be a promising candidate to be used against bacterial pathogens.

This study was conducted to survey the antimicrobial activity of Lactococcus lactis subsp. cremoris NABRII66 lactic acid bacterial strain against common bacterial pathogens in aquaculture and human populations by in vitro experiments. L. lactis subsp. cremoris NABRII66 has been isolated from rainbow trout intestine. Two methods including double layer agar and microtiter plate assay were used to evaluate the inhibition activity of selected bacterial strain against two bacterial pathogens L. garvieae and Aeromonas salmonicida species affecting salmonids and other fish speciesand two other bacterial pathogens including Escherichia coli and Staphylococcus aureus, as two most common source of infection in human population. The results of double layer agar method showed the antimicrobial activity of L. lactis subsp. cremoris NABRII66 bacterial strain against all the tested pathogens and the highest significant growth inhibition zone was observed against A. salmonicida and S. aureus bacterial pathogens (p<0/05). The results of the microtiter plate assay method showed that cell-free supernatant of L. lactis subsp. cremoris NABRII66 bacterial strain could significantly inhibit the growth of A. salmonicida (p<0/05). This study showed that Lactococcus lactis subsp. cremoris NABRII66 bacterial strain isolated from rainbow trout intestine had antimicrobial activity against four aquaculture and human's bacterial pathogens by producing some extracellular inhibitory compounds, which should be evaluated in future studies.

The purpose of this study was to investigate the effects of the use of three lactobacillus strains (L. reuteri ABRIG17 (MF686477), L. reuteri ABRIG23 (MF686483), L. reuteri ABRIG3 (MF686463)), Isolated from duodenum and Jejunum sections of the native Guilan chickens and one isolate of L. salivarius NABRII58 (MH595986) isolated from the digestive system of the native ducks of Mazandaran, on serum lipids and immune parameters of broiler chickens. The strains were isolated from 383 gram-positive and catalase negative lactic acid bacteria in a screening procedure for the detection of bacteria with probiotic potential. In the experiment, 500 male Ross 308 male broiler chicks were used in a completely randomized design with 5 treatments, 5 replicates and 20 birds per replicate. The treatments consisted of: 1-The basal diet as control group (treatment C), 2- The basal diet + 1 g / kg of mixed powder containing MF686463 (L. Reuteri ABRIG3 (LR1), 3- The base diet + 1 g /kg of L. Reuteri ABRIG23 (MF686483) (LR2 treatment), 4- The basal diet + 1 g / kg of L. Reuteri (MF686477) ABRIG17 (LR3 treatment) and 5- The basal diet + 1 g / kg of L. Salivarius (MH595986) NABRII58 (Treatment LS). The use of bacterial strains in LR1 and LS treatments resulted in improved weight gain at the end of the experiment (P <0.05). The bacterial strains in LR1 treatment improved daily weight gain in whole experimental period (P <0.05). All three strains of Lactobacillus used, namely, LR1, LR2 and LR3 treatments, caused a significant decrease in abdominal fat pad (P <0.05). In LR3 treatment, the increase in carcass weight was observed (P <0.05). Measuring serum immunoglobulins in broiler chicks after two stages of sheep red blood cell injection showed that LR3 had the highest total immunoglobulin level after the second injection (P <0.05), and IgG levels in the LR1 treatment increased after the first injection compared to the control group (P <0.05). After the second injection, the IgG of all the four experimental groups were higher than control group (P <0.05), but IgM showed no significant difference between experimental treatments and control group. Total serum cholesterol concentration of LR2 treatment was significantly (P <0.05) lower than other treatments. There was no significant difference in serum triglyceride level between control and the LR1, LR2 and LS treatments, and only LR3 triglyceride level was higher than others (P <0.05). The level of HDL or good cholesterol in LS treatment was higher than the control group (P <0.05). Serum LDL level did not show any significant difference between the experimental and control groups. The present study revealed that probiotic bacteria can be isolated from native poultry microbial populations and isolated bacteria can improve the production and immunity of broiler chicks. It is worthy of note that the positive effect of Lactobacillus salivarius strain isolated from the native duck gastrointestinal tract on the weight gain and serum immunoglobulins in broiler chickens suggested that the gastrointestinal bacteria of a species of poultry could be used as a probiotic in other types of poultry.

In the recent years, there is evidence of training a red type of zebrafish which differs from wild-type in body color. There is not any document how it reaches to the ornamental fish farms of Iran but at first, it was a doubt it belongs to a morphotype or genetic modification (GM). First of all, a set primer was designed to validate zebrafish species. Mitochondrial 16srDNA was selected and amplified. The validation of 16S rDNA gene sequences data detected three haplotypes deposited in the GenBank. By seeking scientific articles about transgenic zebrafish, the first attention went to Glowfish (color transgenic zebrafish). We decided to build a foundation of PCR based detection for finding a gene correspondence red color of zebrafish. This sequence is specific for producing a red color in the sea anemone and it was not in genome database of wild zebrafish. A set primer was designed according to the red color of sea anemone (dsRed) to make a fragment of 680 bp in red zebrafish and cream zebrafish specimens (they had red parents). Fragment sequencing (ds/Red) revealed that it belongs to the sea anemone and none of the samples nucleotides differ from each order and positive sequence from a plasmid containing dsRed. Also, protein samples from cream and red zebrafish were extracted and evaluated with fluorescence microscope by TxRed control color. A dark and a red spread was detected for protein smear of the cream and redfish respectively. A dark spread was detected for cream fish protein smear but a red smear was marked for redfish protein. This research simply demonstrates the existence of transgenic zebrafish in Iranian ornamental fish industry.

Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8th week of pregnancy, by maternal blood sample testing. The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method. Maternal blood samples were taken from 40 pregnant cows during the 8th-38th weeks of gestation. DNA was extracted from 350 µl of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A260 and purity (A260/A280) of extracted DNA were detected by ultraviolet spectrophotometer. Three µl of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes. The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 (p>0.05, p=0.3549), but the difference between mean purity (A260/A280) of DNA extracted by phenol-chloroform method and salting-out method was significant (p<0.001). X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results. The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma.

Bovine growth hormone(bGH) gene, is a part of the multiple gene family that contains prolactin and placental lactogens. The variations in the introns of this gene, have potential usefulness as genetic markers and could help in the genetic improvement of populations. In ordre to investigate allele frequency of MspI in introne 3 of growth hormone gene of Guilan native cattle breed, blood samples were collected from seventy heads of randomaly choosen cattles (cows). Genomic DNA was extracted from these samples, using modified salting out method. Polymerase chain-reaction (PCR) procedure was used to amplify a 345 bp segment in this region and then digested with MspI restriction enzymes. Digested samples were run on the acrylamid gel 8% to recognize their genotype. This enzyme had two restriction sites and after digestion with MspI enzyme on that fragment and 2 allels and 3 genotypes have been observed. Frequency of MspI(+) allele was estimated to 0.4643. X2 test has showed the equilibrium of frequency MspI in samples.

In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction (PCR) analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 μl maternal plasma. Two primer pairs for bovine amelogenin gene (bAML) and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses.