RESUMEN
Shigella, causative of bacillary dysentery, has two colony forms. The loss of large virulence plasmid from virulent Shigella sonnei form I, during cell storage and subculturing, lead to avirulent form II. Environmental factors, e.g. culture media composition, could affect the conversion of the bacterial forms. In this study, some components, i.e., B-complex vitamins, nicotinic acid and riboflavin, were added to the bacterial culture medium and their influence on colony conversion were examined. The findings revealed that colony conversion is temperature independent and growth on the SS agar did not stabilize the bacterium in form I. Also, the findings showed that colonies on the minimal media supplemented with nicotinic acid and riboflavin, were stable in form I. In addition, according to the findings, the active OxyR has potential binding sites upstream of two genes involved in the replication of large virulence plasmid and expression of O-polysaccharide, i.e., repB and wbgT, respectively. Based on the findings of the present study, it is possible that nicotinic acid and riboflavin activate the transcriptional regulatory protein OxyR via dropping off the intracellular reducing power and in this way stabilize the colonies in form I
RESUMEN
Objective: this study was carried out for identification of Eimeria spp isolated from poultry breeder farms in Iran by PCR
Design: pop-Gene Analysis
Animals: poultry breeder farms
Procedures: a total of 114 litter samples from poultry breeder farms without previous exposure to anticoccidial vaccine were collected randomly from relatively five different climate regions of Iran. DNA was extracted from oocysts of samples, using phenol- chloroform and proteinase-K. Four pairs of specific primers, designated from Internal Transcribed Spacer-l[ITSI] regions of ribosomal DNA of E. acervulina, E. brunetti, E. necatrix, and E. tend/a and one pairs of universal primer BSEF-BSER which amplify ITS 1 of different Eimeria were used in PCR assay. In tests on purified genomic DNA from all species of Eimeria isolated from infected samples, each of four primer pairs amplified the ITS! region of their respective target species only. The PCR products were analyzed by agarose gel electrophoresis
Results: DNA fragments in sizes of 320 pairs [E. acervulina], 311 pairs [E. brunette], 384 pairs [E. necatrix] and 287 pairs [E. tenella] were detected on agarose gel electrophoresis. Universal primer pairs also amplified ITS1 of five Eimeria which isolated from infected samples
Laboratory implications: the results of this study were showed that PCR technique is a conventional method, faster, technically easier and very cheaper than other methods to identify the Eimeria spp. Finally, this technique can be recommended to be a routine work in well equipped veterinary diagnostic labs in IRAN
RESUMEN
Rotavirus non structural glycoprotein NSP4 can induce diarrhea in newborn mice. It has been suggested that NSP4 may be a key determinant for rotavirus pathogenesis and a target for vaccine development. In order to study the biological and morphological role of NSP4 a large amount of the purified protein and antibody against it are required. Simian rotavirus SA11 was propagated in BSCI cell, purified on cesium chloride gradients, and its genomic RNA was extracted. A cDNA from RNA segment 10 was synthesized and amplified by RT-PCR. cDNA fragment was cloned into plasmid vectors. The recombinant plasmid was characterized by restriction enzyme and sequencing. Construction of expression plasmid containing nsp4 gene was performed and expression of NSP4 was demonstrated by SDS-PAGE, Western blot, ELISA and IF using polyclonal antibody against NSP4 from SA11 infected BSC1 cells. A polyclonal antiserum against purified recombinant NSP4 was raised in rabbit; which was reacted with NSP4 in BSCI cells infected with SA11 rotavirus. These results indicated successful expression of the full-length NSP4 in E.coli to produce antibody against it and to study its biological activities