جستجوی مقالات مرتبط با کلیدواژه « rt » در نشریات گروه « دامپزشکی »

Leptin, the product of the obesity (ob) gene, acts as a signaling adipokine for modulating food intake, energy metabolism and reproductive functions in mammals. Leptin’s effects on the reproductive system at various levels of the hypothalamic-pituitary-gonadal axis have been established. Moreover, the direct and local effect of leptin on bovine oocyte maturation and corpus luteum function has been determined.
Due to species differences, this study was designed to investigate expression of leptin mRNA as well as its long isoform receptor (Ob-Rb) mRNA in sheep corpus luteum.
Ovaries of sheep containing mature corpus luteum were collected in the reproductive season from abattoirs. Total RNA of corpus luteum was extracted, cDNA synthesis was carried out subsequently and PCR reaction was performed using primers which were designed specifically for each gene. Beta-actin was used as housekeeping gene to verify reactions, and adipose tissue was selected as positive sample for expression of leptin and leptin receptor.
Gel electrophoresis of PCR products showed the amplification of 162 and 121-bp amplicons in all samples for leptin and leptin receptor respectively. Moreover, sequencing the amplified fragments and blasting them confirmed the accuracy of results.
Our findings confirm the expression of leptin and its functional receptor transcripts in ovine corpus luteum. More studies for determining leptin effects on corpus luteum are guaranteed.

FMD is one of the most important animal health problems in the world and is ranked at the top of the list A of potentially epidemic infectious diseases of livestock (OIE). FMD virus infects a wide range of domestic and wild cloven hooved animals and causes clinical signs. The disease is mild zoonotic and 70 wild mammal species from 20 animal families are susceptible to infection. Also, birds are mentioned as transferring agent of FMD virus in several references.
The motivation of this study was due to observation of a significant presence of pigeons in FMD involved farms in the epidemic of serotype O2016 in the first months of 2016.
After hunting of six pigeons gathering food in FMD involved farms, their blood samples were collected. In the laboratory, FMDV genome was traced by RT-PCR with aphtovirus universal primers and final product was sequenced.
The 328 bp band indicating a positive result was observed in electrophoresis of all samples. These results were also confirmed in repeated experiments. Then the RT-PCR products were sequenced in both directions. Alignment and BLAST results indicated more than 97% identity of virus from samples with FMD registered viruses in Genebank, demonstrating the presence of FMD virus genome in the blood of the pigeons.
This result indicates FMD virus genome viremia in the blood of pigeons. It is worth noting that pigeons’ infection is very important because this species is a free flight bird and has the possibility of transmitting the virus over long distances, thereby causing new epidemics. Finally, it is necessary for further studies to investigate the possible presence of clinical signs in the pigeons, the possibility of shedding, routs and virus titers of shedding from any of the possible ways.

Cloned vaccines are used in many countries nowadays. One of the ways for cloning a virus is propagation of the virus on cell culture to obtain discrete different plaques in order to study their morphology and genetics. In this study monolayer Madin-Darby Canine Kidney (MDCK) cell cultures were prepared by standard method. Various dilutions of the viruses were inoculated into monolayer MDCK cell cultures that were supplemented with magnesium sulfate and trypsin, and over laid with agar medium. The viruses could reproduce on these cells and caused cytopathic effect and plaques. At 10-6 virus dilution, 6 various shape and size discrete plaques were obtained and inoculated into allantoic fluid 9-11 days embryonated eggs. After 48 hrs, the allantoic fluids contain plaques were harvested and their RNA extracted. Cleavage site of fusion protein, with RT-PCR test was performed and the PCR products were purified and sequenced. The sequences of nucleotides and amino acids for each plaque were compared with those of the registered strain at gene bank as well as with each other. Molecular studies showed that all plaques are lentogenic strain of Newcastle disease virus and has about 97% to 99% homology with the strain V4 in the gene bank. The aim of this study is produce clear plaque by V4 strain of NDV on MDCK cell line and studies the molecular variations among them.

Newcastle disease is common in broiler farms. It causes huge losses to the poultry industry. Because of these, investigating and identifying the disease are important. Moreover, it would be much better to use simpler and cheaper techniques while being more accurate and more sensitive. Therefore, 30 broiler farms, located in Khuzestan province, were sampled and the suspensions were prepared. Then the suspensions were inoculated into 9 to 11 days oldembryonated chicken eggs and after the incubation period were investigated with hemagglutination test. Twelve samples were positive in the haemagglutination test. Finally RT-PCR was performed on the 12 samples to detect Newcastle Disease Virus and the results were positive in six cases. It is important to mention that all six isolates were brain-sourced (also isolated from other tissues) and since all available Newcastle disease vaccines in Iran are lentogen, so the isolates were not from vaccine virus.

Newcastle disease is one of the most serious viral diseases in the poultry worldwide.

Since the traditional strategies have been hardly effective in controlling the disease, the purpose of this study was to introduce new methods for early and rapid diagnosis of Newcastle. The present study helps to reduce further damage to the poultry industry.

RNA extraction was performed, using RNease mini kit, according to the manufacturer’s instructions. Extracted RNA with 68.23×109 copy numbers was prepared as serial dilutions of 100 μL for RT-PCR and RRT-PCR reactions. RRT-PCR and RT-PCR were performed, using commercial kit and RNease mini kit, respectively.

Results showed that amplification was done according to prepared dilution equal 10-34 for RRT-PCR reaction and a visible band observed on 1.5% Agarose gel up to 10-20 for RT-PCR reaction. Based on the results observed, RRT-PCR and RT-PCR reactions are able to detect 10-34 and 10-20 copy numbers of primary sample, respectively.

The sensitivity of RRT-PCR reaction is almost twice compared with RT-PCR reaction, also RRT-PCR reaction is able to diagnose Newcastle disease virus in infected samples with 10,000 copy numbers of the RNA virus less than RT-PCR.

Avian infectious bronchitis (IB) is caused by corona viruses. Infectious bronchitis virus (IBV) is a major cause of economic losses in poultry and can be involved in respiratory disease، nephritis، and both poor egg production and quality by reproductive tract infection. IBV has many serotypes that do not confer cross protection against each other. The study in 2012 was conducted to detect IBV in broilers chicken farms suspected of respiratory syndrome with a high mortality and no history of vaccination against of the IBV in Charmahal va Bakhtiyari province. In this study، one hundred serum samples and one hundred tracheal and lung tissue samples were collected from 10 broiler chicken farms with respiratory signs. To achieve this goal، two tests have been used throughout the studies of protocol Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The presence of IBV antibodies was detected by using ELISA kit. The results indicated that 7 flocks (70%) were positive and 3 flocks (30%) were negative tested with RT- PCR method. The evaluation of serum antibody titers of flocks showed that antibodies against infectious bronchitis virus in RT-PCR positive flocks were higher than RT-PCR negative flocks. The results also evinced that the prevalence of infection bronchitis in broiler flocks was high in broilers farms of Chaharmahal va bakhtiyari province and concurrent Infection of IBV and other respiratory pathogen played a role in high mortality rate of respiratory disease.

Detection of bluetongue virus in aborted lamb fetuses in Chaharmahal va Bakhtiari and Isfahan provinces، by RT-PCR method Bluetongue or muzzle sore disease is an infectious disease which is mainly transmitted by insects. It can cause erosions in mouth، nasal mucosa and limbs and also abortion in ruminants. The aim of this study was genomic detection of bluetongue virus in sheep aborted fetuses as an aborting agent. Samples were collected from blood، liver and spleen of 50 aborted lambs. EDTA was added to blood samples. RNA was extracted by using Rimazol. cDNA was synthesize was detected by RT-PCR. All products were separated by 1/5% agarose gel electrophoresis and stained in ethidium bromide. The results of all samples were negative، except one of them which was sent for sequencing and the result of sequencing didn''t confirm presence of this virus. This study shows that Blue tongue virus does not cause abortion in ewes in studied areas of Iran، but it is possible that by increasing the sample size and sampling during the year in these areas، the presence of virus or its genome can be detected too.

In this case-study، geographical distribution of infectious pancreatic necrosis (IPN) was studied in 18 provinces of Iran using RT-PCR during 2011-2012. Since the occurrence of coinfection by IPNV، infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) with the same clinical signs can occur، all samples were tested for presence of these three virus agents. Samples of larva and juveniles from rainbow trout with clinical signs such as anorexia، exophthalmia; darkened skin and high mortality as well as sperms and oval samples were obtained. Results showed that from 214 pooled samples، 96 (44. 85%) in 15 provinces were positive for IPN. The highest relative frequency was observed in Lorestan، Isfahan، Chaharmahal va Bakhtiyari، Fars، Kermanshah، Kohgilouye va Boyerahmad، Mazandaran and Qazvin. These results show that IPN disease is distributed in north، western north، west، western south and south parts of country. Therefore it is important to improve the preventive criteria to decrease the economical losses due to the disease morbidity and mortality in farmed trout in Iran.

Feline calicivirus (FCV) is a highly infectious respiratory pathogen of domestic cats with a widespread distribution. In order to assess how FCV circulates in feral and household cats، we have carried out the first study on the FCV detection in Ahvaz district from December 2008 to November 2009. Oropharyngeal، nasal and ocular swabs of one hundred cats (70 feral and 30 household) were evaluated by reverse transcription polymerase chain reaction (RT-PCR) procedure for the detection of FCV. The influence of sex، age، social status and clinical signs on the probability of infection was analyzed using statistical Fisher’s exact test. Overall، feline calicivirus was detected in 4/100 (4%) of sampled cats; 13. 3% of the household cats were FCV positive compared to 0% of feral cats. According to several factors including younger cats (under 6 months of age)، multiple cat household and clinical findings، differences were significant (p<0. 05) in statistical analysis. There was no significant difference between the sex distributions of the cats (p>0. 05). To the best of our knowledge، this is the first report indicating the presence of FCV in cats in Iran. Due to low prevalence of FCV infection and the fact that feral cats live solitarily، it was concluded that this viral infection don’t spread readily within feral populations. However special measures are recommended to avoid infection of susceptible and unvaccinated cats.

Genetic variations in the hemagglutinin gene of influenza viruses are an important characteristic. These variations may cause limitation of RT-PCR assay for detection of influenza subtypes. In this study, a total of 6 samples, suspected to type A influenza virus, were isolated from industrial birds’ trachea in North Khorasan Province and RT-PCR analysis were performed. Results showed that all of the 6 suspected samples were detected positive as type A influenza viruses. These isolates were then determined their subtypes using RT-PCR H9N2 specific primers. The result showed that 4 out of 6 isolates were positive as H9N2 and other two isolates did not have any bond on gel electrophoresis and considered negative. For further evaluation, all of the hemagglutinin genes of viruses were sequenced and confirmed as H9N2 viruses using BLAST software in Gene Bank. It was also found, that primers binding sites with hemagglutinin genes in two last viruses were mismatched in three nucleotide acids with 3' end of forward and reverse primers. These factors may reduce the sensitivity of RT-PCR tests and cause false negative results. Therefore, it would recommend updating RT-PCR primers and protocols in a timely manner.

Avian influenza virus has been recognized as one of the most important pathogen in poultry that caused economic losses to the poultry industry of Iran. Rapid detection and pathotyping is the important priority in order to prevent extension of disease in Iran. RT-PCR method was used for the rapid detection of influenza viruses. The amino acid sequence of cleavage site of hemagglutinin was considered as a molecular determinant for the prediction of pathogenicity influenza viruses. In this study، 160 tracheal swabs samples were taken from 20 poultry farm to monitor influenza infection in North Khorasan province. RT-PCR assay was developed based on the most common genotype in Iran detecting type A and H9، H5 and H7 subtypes by specific primers targeting Matrix (M) and Hemagglutinin (HA) genes، respectively. The samples which were positive as type A influenza inoculated to embryonated eggs of 9-10 days. After 3-5 days، the allantoic fluids were tested with Hemagglutinin assay. The samples that contained HA activity were inactivated with formalin 0. 1%. These fluids were used for RNA extraction. A segment of 432bp was amplified by RT-PCR and nucleotide sequencing. The results showed that four poultry farms were positive for type A of influenza virus. All of them were negative for H5 or H7 but positive for H9 virus. The sequence analysis of hemagglutinin revealed that four isolates shared a KSSR motif at the cleavage site of HA. Although this motif represented low pathogenicity in poultry، it was different from previous Iranian isolates. As a result RT-PCR assay based on common genotype in region was considered as a rapid test for the detection of influenza viruses. The cleavage site sequence analyses indicated that these viruses have the potential for emerging highly pathogen influenza viruses. Therefore، continuous monitoring of viruses is being recommended.

Considering the lack of knowledge of the molecular biology of scorpion venom antibacterial peptides، as a first step we sequenced and identified a full-length sequence from the venom glands of scorpion (Mesobuthus eupeus) through RT-PCR. Scorpion toxin consists of different types of toxins and enzymes which are encoded by individual genes. Antimicrobial peptides are cytolytic peptides that can rapidly kill a broad range of microbes and have additional activities that impact on the quality and effectiveness of innate responses and inflammation. These peptides had been found from a wide variety of organisms in the last few years. Scorpions were provided and identified by Razi Institute in Ahvaz. The origin of the total RNA was the telson، the last segment of the tail containing the two venom glands. Using RNX plus solution (CinnaGen، Iran)، the total RNA was extracted according to the manufacturer’s instructions. Then، cDNA was synthesized with the extracted total RNA as template and oligo (dT) as primer. In order to amplify cDNA encoding neurotoxin، RT-PCR was performed. The amplified cDNA fragments were sequenced in both strands according to the dideoxy termination method. Sequence comparisons with GenBank database were done using the BLAST software. Based on the sequence data، the complete sequence contained 273 nucleotides called MeBTXA signal peptide sequence was identified، and the glycine at position 20 was assumed to represent the start of the mature protein. Remarkably، they share 96، 94 and 67% similarity with the long-chain potassium ion channel blocker from Mesobuthus martensii، Buthus occitanus Israelis and Tityus costatus. Domain analysis of the protein showed «Arthropod defensin» between amino acid resides 55 to 85. This domain which is rich in cysteine is belongs to the antibacterial peptide family and has effect on the gram positive bacteria. This high sequence similarity together with completely conserved in the positions of all 6 cysteine indicated that this gene is a new member of β-toxin from the Iranian M. eupeus which may originate from a common ancestor.

The bovine viral diarrhea virus (BVDV) is one of the bovine respiratory system pathogens that represses lungs immunity and raises the pathogenicity of other bacteria and viruses. The aim of the present study was to evaluate the effect of BVDV in lung tissue. In this study, 30 bovine lung tissues were used. Histopathologic analysis showed different lesions in lung tissues. RT-PCR test results and histopathologic observations were studied for each sample and compared. Kai Square Test was used to study any possible association between the existence of BVD virus and incidence of four common lesions: 1- Interestitial pneumonia, 2- bronchitis and bronchiolitis, 3- edema, emphysema and atelectasis and 4- bronchopneumonia. If the assumptions of X2 test were not applicable, the Fisher Exact Test was used. The RT-PCR test for BVDV results was positive for 10 lung samples. 5 BVD positive cases were involved with interestitial pneumonia, while 10 BVD negative cases were not affected. Finally, in the studied lung tissues, only the 3 + degree or severe interstitial pneumonia was significantly associated with the existence of BVD virus. No significant relation was seen regarding other possible lesions, which may have resulted from the probable roles of bacteria or other viruses and toxic factors that were not studied in this project.

Broilers lung mechanisms that regulate endothelin (ET) in the lung are complex and poorly understood. Objectives/ In this experiment lung ET-1 mRNA levels and lung mRNA expression for the ET(A) receptors were determined in lung tissue weekly (term = 42 days, intervals = 7 days). Serum endothelin concentration was also measured at these ages. The study showed that expression of endothelin in lungs of layers and broilers was similar during the first three weeks and the overall trend of ET-1 expression was increasing. However, there was a significant increase of ET-1 expression which started from the fourth week and gradually increased until the end of the commercial life of the chicken (day 42). ET-A expression in the lungs of broilers was significantly higher than layers during the last three weeks of life. Overall, trends of serum ET-1 concentration increased in both layers and broilers, but serum ET-1 concentration in broilers was significantly higher than layers. The higher level of serum Endothelin and expression of ET-1 and ETA in broiler lungs may explain the higher sensitivity of broilers to the vasoconstrictions activity of endothelin and the higher sensitivity of these animals to pulmonary hypertension (PH).