Development and ELISA-based detection of anti-M2e IgY antibodies using an encoding plasmid for M2e-Hsp70 c-terminal gene

The use of IgYs in a variety of methods in different areas of research, diagnostics, medical application and biotechnology should be considered widely. Development of antibodies against extra cellular domain of influenza M2 [M2e] protein in egg yolk of laying hens. A Fusion construct harboring C-terminal of bovine heat shock protein 70 [Hsp70] and influenza M2e coding genes was injected to laying hens. Serum and egg yolk antibodies were screened for the presence of anti-M2e antibodies by indirect enzyme-linked immunosorbent assay [ELISA]. Anti-M2e antibodies were detected in egg yolks and sera of injected hens from 13 and 7 days post injection [PI], with the peak titer detected on 41 and 35 days PI, respectively. Anti-M2e IgY titers could be an index for expression potential of pcDNA3.1-M2e-HspCterminal construct in laying hens. This construct could be considered as a promising tool in production of anti-M2e polyclonal, monospecific IgY antibodies. Such anti-M2e antibodies could be exploited for influenza diagnostic and therapeutic measures

[Cloning, sequencing and expression of HSP70 c-terminal domain in Mycobacterium avium subsp. paratuberculosis]

Heat shock proteins [HSPs] have been shown to act as an adjuvant when co-administered with different antigens, especially tumor antigens or antigens from infectious agents. C-terminal domain of Mycobacterium tuberculosis heat shock protein 70 [Hsp70], when fused to peptide antigens, provides a unique structure that is able to induce potent immune responses. In this study, aneukaryotic expression vector pEGFP-N1, containing C-terminal domain of Mycobacterium paratuberculosis HSP 70, Green Fluorescent Protein [GFP] gene in the plasmid construct, was designed for use as a reporter. With GFP system, expression of the target protein was evaluated in the cell culture. The nucleotide sequence of the cloned gene was revealed by sequencing. The protein expression of designed plasmid was also proved by reverse transcriptase polymerase chain reaction [RT-PCR]. Our eukaryotic expression vector [pEGFP-N1 -hsp70 c-terminal] was successfully constructed and HSP70 c-terminal domain protein was expressed effectively. The current experiment, as a basis for a new DNAvaccine design, can be used for the future studies on reverse vaccinology

[Association between amino acid sequences in peptide binding region [BOLA-DRB3] and susceptibility or resistance to calf diarrhea]

The major histocompatibility complex genes [mhc] encode MHC I and II molecules which present peptide fragments to T cells. Therefore these polymorphic molecules critically influence susceptibility to infectious diseases. At present study potential relationship between amino acid sequences in the antigen binding groove of different BoLA-DRB3 alleles and susceptibility or resistance to calf diarrhea was investigated. Twelve different DRB3 alleles were found among 171 calves [84 diarrheic and 87 healthy] analyesd by PCR-RFLP method. Amino acid sequences of the encoded peptide binding region were compared. 26 polymorphic positions were detected in this region. A significant association [p<0.05] was shown between occurrence of diarrhea and the presence of glutamic acid and tyrosine inpocket 4 and valine, glutamine and leucine in pocket 9 of peptide binding region. Thus it can be concluded that pockets 4 and 9 of the BoLA-DRB3 molecule would be involved in conferring susceptibility of calf to diarrhea

[Identification of mycobacterium avium subsp paratuberculosis in fecal samples of holstein-friesian cattle using molecular and cultivation methods]

Mycohacterium avium subspecies of Paratuberculosis is the cause of paratuberculosis or Johne's disease. Paratuherculosis is a chronic and progressive infectious enteric disease that affects domestic and wild animals, mostly ruminants. This study was carried out for detection of subclinical forms of Johne's disease using direct PCR and cultivation methods. A total 119 fecal samples were collected Holstein-Friesian cattle herds in Razavi khorasan province of Iran. Among these, 16 samples were taken from cows with advanced clinical signs of Johne's disease. 101 were taken from cows without clinical signs. The fecal samples were tested for presence of Mycobacterium paratuberculosis by directed Nested-PCR and culture on Herrold s media with and without Mycobactin J. The samples of cows with clinical signs, 14[87.5%] and 13[81.3%] were positive by PCR and cultivation assays, respectively. These numberes for the 101 cows without clonical signs of Johne's disease were 10[9.7%] and 12[11.7%]. In conclusion, according to the current report of bacterial detection from different places and the economic importance of John's disease, it is logical and essential that the prevalence rate of the disease in dairy cattle is initially determined by at least two different types of samples and tests and afterwards the control programs are adopted

Cloning and high level expression of bovine interferon gamma gene in eukaryotic cells [COS-7]

Interferon gamma [IFN-gamma] is one of the key cytokines in defining T helper 1 lymphocyte immune responses. In this study, the bovine IFN-gamma gene was cloned from spleen tissue RNA using the reverse transcription-polymerase chain reaction [RT-PCR]. IFN-gamma cDNA was sub-cloned and expressed in mammalian expression plasmid [pcDNA3.1[+]] under the control of the human cytomegalovirus [CMV] promoter. The predicted amino acid [aa] sequence of bovine IFN-gamma compared with corresponding known sequence from bovine [Bos taurus] was 100% identity and with ovine, caprine, camel, lama, equine, canine, feline, human, mice and chicken cytokine was 95, 95, 86, 83, 77, 75, 75, 61, 44 and 35%, respectively. Invitro expression of recombinant bovine IFN-gamma [rBoIFN-gamma] and secretion to culture medium was confirmed by ELISA test. Maximum expression of rBoIFN-gamma occurred at 96 and 144 h after transfection in COS-7 cells. These results showed that pcDNA3.1 expression vector and COS-7 cells transfected by diethylaminoethyl [DEAE]-dextran allowed the high level expression of bovine IFN-gamma gene and the release of protein in supernatant of cell culture

[aroA gene sequencing of native Escherichia coli O78: k80 isolated from avian colibacillosis in Iran]

aroA is an important gene which produces aromatic amino acids essential for bacterial life. The sequence of this gene was shown to be different in bacteria, so the purpose of this study was sequencing of aroA gene in native E. coli O78: K80 to determine the putative differences between this strain and other E. coli O78: K80 strains in gene bank. PCR was used to amplify aroA gene in native E. coli O78: K80. The amplified 1.206 kb PCR product was extracted from Agaros gel, ligated in pTZ57R and sequenced. Blast analysis showed that aroA sequence in native E. coli is different from already submitted homolog gene in Gene Bank. Two amino acids were shown to be different from those already found in other E. coli strains. This different amino acids are Isoleucine and Glycine instead of 39[th] Threonine and 240[th] Glutamic acid, respectively. Knowing these differences are important for doing molecular techniques, designing primers and future studies

Application of combination, immunomagnetic separation plus multiplex PCR for the detection and identification of salmonella enterica subspecies enterica serovar typhimurium in bovine diarrhoeic fecal samples

A total of 400 bovine diarrhoeic fecal specimens were obtained and conventional microbial culture, immunomagnetic separation and multiplex PCR were simultaneously carried out on samples. For detection of Salmonella at genus level, inv-A universal primer was selected. In order to identifiing of Salmonella typhimurium, specific primers of Rfbj, Fljb and Flic related to gene sequence of O4, H2:1,2 and H1: i were used, respectively. Results showed, 33[8.5%]were culture positive for Salmonella serotypes. However, Salmonella typhimurium with[66.7%], Salmonella dublin[9.1%], Salmonella virchow[6.1%], Salmonella gloucester[6.1%], Salmonella enteritidis[3%], Salmonella georgia[3%], Salmonella augustenborg[3%]and Salmonella lindenburg[3%], were the most common isolated serovars, respectively. In the IMS+Multiplex PCR four amplified products[663,526,284 and 183 bp] were found in all specimens which had typhimurium serovar[1,4,5,12:i:1,2]from rfbj,fljb,inv-A and flic genes, respectively. Results showed that detection and identification of Salmonella typhimurium using specific primers of O4, H2:1,2 and H1: i antigens can be useful

Rabies in the course of iranian history

On the rabies and a rabid dog, Iranian physicians have said and written much and they have introduced their innovations now and then. Kharazmi in his Mafatih al- Olum [Keys to Sciences, written in 980 A.D.], writes on rabid dog: "Amad dog is called a rabid dog which bites, avoids eating and fears water and dies in deep thirst. If such a dog bites a human being, he will be inflicted with a dangerous disease which makes him fear water [Hydrophobia]. Rhazes says: "It does not recognize its owner, and attacks whatever on his way. Its mouth is always open with its tong hanging out. Eyes are red and other dogs run from it." [Al- Havi]. Avicenna, Jorjani, Youssef ibn Muhammad Heravi and other grand physicians of Iran have written intresting points about the symptoms, incubation period and treatment of rabies in man and dog, differences between a rabid dog and a biting dog, rabies in wild beasts, and the like. Avicenna wrote in canon:" It is said and approved by many physicians that the liver of a rabid dog if is kept on the wound of the bitten person will be helpful". Also he said clearly: "The blood of the rabid dog is antitoxin for rabies." This is a kind of serotherapy or vaccination. The liver of a rabid dog contains various antibodies against rabies, and if it is applied to a recent bite wound, some antibodies may enter the wound. Therefore, the procedure might conceivably be effective in preventing rabies. This is a matter of serotherapy and immunology that was introduced more than 900 years ago

Sayyed Esma'il Jorjani, founder of Persian medicine

Sayyed Esmail Josjani [Gorgani] was one of the greatest scientists and physicians of Iran died in 1136 in merve. He promted writing medical book in Persian by authoring the valuable book of zakhireh kharazmshahi and Al Aghraz al tibia. Of course writing medical books in Persian was common before Avicennas time [980 - 1037]. Zakhireh and Aghraz are like big and medium medical encyclopedia and even since the creation of the present Persian script. These books are the last and most important medical books in Persian. Like any other ancient book, some parts of these books are abolished but some other parts are valuable. However these books are remained to be unique as Persian medical books in the course of time. I have edited and corrected al - Aghraz al tibia [Medical goals] from several old manuscripts. There are seveval manuscripts of al - Aghraz [which is about 250000 words]. To edit and correct this book, I have used five old important manuscripts of Aghraz and one manuscripts of zakhireh kharazmihahi. I have published this book in 2 volumes in 2005 - 2006. At the end of the second volume, I compiled a medical dictionary named Dictionary of Aghraz. The references of this work are various documents including manuscripts of published, old and new medical sources in persain or Arabic languages and some English or French documents

Cultivating people for preventing environmental destruction

The fundamental elements of nature, namely earth, water, air and sunshine should be in a healthy state, well balanced with the criteria of equilibrium and untainted so that the environment would remain pleasant and lively. Hence, there will be plant, animal and human in a existential balance. Man himself is the major polluter and destructor of environment. The environment, in turn, is a large collection of elements and a variety of active and reactive factors, developed and evolved for millions of years. As we are witnessing, any disturbance and destruction of nature has unrepiarable and harmful consequences. The protection of environment from man's disturbance calls for formulating appropriate regulations and rules and their right and timely enforcement. Yet, it is more important to cultivate the protection of environment in each and every individual including the laypeople and authorities. If we do not believe in what we are doing, we will fail to do what we are obliged to do. In the Holy Quran, frequent mentions have been made of the prostration of the earth, heaven, mountains, plants, animals and whatever exists in the universe. A type of spirit and expressiveness exists in everything. In other words, the secret of life is hidden in the living and the lifeless. Be it as it may, we should not disturb and destroy none of these things, neither should we find theoretical or artificial justification for the destruction of environment. Otherwise, we will disappoint the present and future generations, and will be responsible for our deeds against the truth. Man continues to strive to make a better and more convenient life for himself and to develop faster vehicles. If these are down properly, they will be useful for him but exploiting the environment is often destructive. The best productivity is to use the resources in a way that it would be less destructive and the environment is untainted minimally and that restoration and reconstruction cycles revive the environment rather than destroying it

Molecular epidemiology of Salmonella Abortusovis in Iran

Study of different genotypes amongS.abortusovis strains. Observation study. 58 Salmonella abortusovis strains belonged todifferent countries were studied. IS200 fingerprinting by Southern blothybridisation have been applied for genotyping. 8 different genotypes identified within strainsunder study. All genotypes contained 3 to 5 copies ofIS200 and revealed high relatedness to their origins. Using IS200 fingerprinting for IranianS.abortusovis strains showed high polymorphism becauseeach province at least had one distinct pattern. Also thismethod remains as a sensitive method for typing theS.abortusovis

Evaluation of Salmonella abortusovis detection by primers related to serovar specific IS200

Evaluation of primers designed due to theserovar specific IS200 copy for detecting Salmonellaabortusovis strains isolated in Iran. Observational study. Ninety seven Salmonella abortusovis strains. PCR amplification was carried out by serovarspecific primers and different strains according to PCRresults were studied by IS200 fingerprinting analysis. All strains could be classified in 2 distinctgenotypes by 2 kb and 900 bp amplicons in PCRamplification. These two genotypes were related to twodifferent profiles with 11 and 9 kb band respectively in IS200 fingerprinting. PCR amplification by serovar specificprimers was capable of grouping the strains in 2 majorgenotypic patterns

[Survey on biochemical characterization and transferable R-factor patterns for biotyping S. abortusovis strains isolated from chaharmahal and bakhtiari province of Iran]

To study blechemichal properties and comparing current antibiotic resistance of S. abortusovis isolated from sheep in Ch-Bakhtiary province and study the prone to R - factor transferability. Comparative study. Thirty four strains isolated from Chaharmahal-Bakhtiary in 1999-2000. Biochemical tests were performed with standard diagnostic tests, antibiotic susceptibility and it's transferability were examined. All strains were resistant to Ampicillin [Am] and Amoxicillin [Amx]. Although the Streptomycin [S] after the Am and Amx was the most common resistance. Resistance to Amicacin [AN] and Tetracyclin [Te] were very low [about 2%]. All strains were susceptible to Chloramphenicol [C], Nalidixic Acid [Na], Kanamicin [K] and Enrofloxacin [En]. Just in one case the profile of Am - Amx - Cotrimaxazole [SXT]- AN- Te wasdeveloped. Among the total isolates, 3 cases were able to transfer some part of their resistance factors. In all three cases only resistance to Ampicillin were transmitted. Antibiotic resistance in S. abortusovis strains isolated from Iran is not that high and rarely were transmited to other bacteria. This phenomenon may be related to the defficalties of drug administration in sheep and got flocks

[Study of Salmonella plasmid virulence genes [spv] in Salmonella enterica serovars isolated in Iran]

Objective: to study the presence of spv genes among different Salmonella serovars that isolated in veterinary microbiology department

Design: observation study

Samples: a total of 138 Salmonella strains belonged to 9 different serovars were studied

Procedure: in this study we applied PCR method using PG44 and PG48 primers to amplify spvR gene in different serotypes

Results: in PCR amplification, serotyps Sabortusovis, S.dublin, S.typhimurium and S.brandburg developed the 890 bp amplicons. S.typhi, S.senfrenberg and S.bovismorbificans have yielded nonspecific bands of different sizes. S.newport revealed no band in amplification

Conclusion: salmonella serotypes such as typhi, senfrenberg and bovismorbiJicans with nonspecific bands in PCR amplification does not share virulence plasmids. Furthermore, spvR loci could be considered as a good marker for presence or absence of virulence plasmids in different Salmonella serotypes

[Comparision of total and cytoplasmic protein pattern in Salmonella enterica subsp. enterica serovar abortusovis strains isolated from Iran]

Objectives: the comparison of total and cytoplamic protein patterns between Salmonella abortusovis strains isolated from Iran

Design: electrophoretic study of protiens

Samples: salmonella abortusovis strains isolated from Iran

Procedure: total proteins were prepared by sonication with a microbrobe. Cytoplasmic proteins were percipitated by the addition of 3 volumes of acetone. Total and cytoplasmic extracted proteins were analysed by SDS-PAGE and silver staining

Results: the results showed that there were considerable differences between total protein profile of S. abortusovis strains. The cytoplasmic protein patterns were similar among the strains

Conclusion: the protein profile differences and number of protein bands are showed in figure and tables. Infact this variations indicating a certain genotypic distance between bacterial strains that isolated from different geographical area of Iran and may be usful for describing the epidemiology or at least genetic relatedness of Salmonella abortusovis wild types