Određivanje P (F11) i F1 fimbrija bakterije Escherichia coli izdvojene iz pilića s avijarnim celulitisom.

Avian cellulitis has been induced by virulent strains of E. coli. The purpose of this study was to determine P (F11) and F1 fimbriae of 90 E. coli isolates of avian cellulitis. Isolates were subjected to six consecutive passages on solid and static broth for expression of fimbriae. Five (5.5%) isolates from O1 serogroup, showed a mannose-resistant haemagglutination MRHA pattern when grown on solid medium. In the SDS-PAGE, crude fimbrial extracts of MRHA strain showed a major fimbrial subunit of 18 kDa. This band was also reacted with anti F11 serum on immunoblotting. Sixty-nine (76.6%) E. coli isolates from different serogroups showed mannose-sensitive hemagglutinating (MSHA) pattern when grown on static broth medium. In immunoblotting test, crude fimbrial extracts of MSHA isolates demonstrated a single band with 17 to 17.5 kDa apparent molecular weight as revealed by absorbed anti-F1A serum. It would appear that avian cellulitis E. coli isolates have F1 and P fimbriae similar to those of colisepticemic E. coli isolates.Avijarni celulitis uzrokuju virulentni sojevi E. coli. Svrha istraživanja bila je ustanoviti P (F11) i F1 fimbrije u 90 izolata E. coli uzročnika avijarnog celulitisa. Izolati su bili šest puta uzastopno pasirani na čvrstoj podlozi i bujonu za rast fimbrija. Pet (5,5%) izolata serološke skupine O bili su manoza-rezistentne hemaglutinacijske aktivnosti (MRHA) uzgojeni na čvrstoj hranjivoj podlozi. Postupkom poliakrilamid gel elektroforeze (SDS-PAGE) sirovi fimbrijski ekstrakti MRHA soja sadržavali su veću fimbrijsku podjedinicu od 18 kDa. Ta je podjedinica također reagirala s protuserumom za F11 u testu imunobloting. Šezdesetdevet (76,6%) izolata E. coli iz različitih seroloških skupina uzgojenih na statičnom hranjivom bujonu sadržavalo je manoza osjetljive hemaglutinacijske uzorke. Imunobloting testom dokazano je da sirovi fimbrijski ekstrakti manoza osjetljivih izolata sadrže jednu podjenicu molekulske mase od 17 do 17,5 kDa što je otkriveno apsorpcijom anti F1A seruma. Čini se da izolati E. coli koji uzrokuju avijarni celulitis imaju F1 i P fimbrije slične onim izolatima E. coli koji uzrokuju koliseptikemiju

Genotyping of Virulence Factors of Uropathogenic Escherichia coli by PCR

Background: Escherichia coli is the most causative agent of urinary tract infections (UTIs). Apart from all human infectious diseases, UTI have a high prevalence and in most cases, Escherichia coli is a dominance bacterium which can cause pyelonephritis and cystitis. The aim of the study was to determine the occurrence of some virulence genes expressing fimbriae, production of hemolysin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of Karaj Shahid Rajaii hospital, showing clinical and laboratory signs of UTI.Materials and Methods: In this investigation we isolated Escherichia coli strains from urine samples of patients with UTI during the period of July to December 2012 and studied them for the presence of the virulence genes by PCR.Results and Conclusion: The most abundant virulence factor in this study was fimH. The prevalence of the virulence factors for fimbriae type 1 (fimH gene), pyelonephritis associated pili (pap gene), S-family adhesions (sfa gene), hemolysin (hly gene) and aerobactin (aer gene), was 73%, 46%, 32%, 47%, 57%, respectively

Evaluation of maternal antibody levels for establishing the vaccination program against Newcastle disease in ostrich chicks

ABSTRACT Newcastle disease virus (NDV) is known as one of the most important endemic viral pathogen for various avian species such as ostrich, in Iran. Therefore, establishing a routine vaccination program against ND in ostrich flocks would be useful in order to reduce the danger of this infection. Newcastle disease occurs among the ostriches and leads to high rate of mortality while most of the losses are among the youngest ones. This experiment was designed to follow up the changes of maternal antibody in ostrich chicks during the first weeks of their life. At this point of view, 700 one day old ostrich chicks were monitored and every seven days interval 10 blood samples were taken regularly and the titers of maternal antibody in their sera were studied. The haemagglutination inhibition (HI) test was used to evaluate the amount of anti-ND antibody. After hatching this study followed up to 49 th day. Due to our findings, the day 30 is recommended as a proper time to start the vaccination program against ND in flocks of ostrich chicks with maternal antibody

Infections with Avian Pathogenic and Fecal Escherichia coli Strains Display Similar Lung Histopathology and Macrophage Apoptosis

The purpose of this study was to compare histopathological changes in the lungs of chickens infected with avian pathogenic (APEC) and avian fecal (Afecal) Escherichia coli strains, and to analyze how the interaction of the bacteria with avian macrophages relates to the outcome of the infection. Chickens were infected intratracheally with three APEC strains, MT78, IMT5155, and UEL17, and one non-pathogenic Afecal strain, IMT5104. The pathogenicity of the strains was assessed by isolating bacteria from lungs, kidneys, and spleens at 24 h post-infection (p.i.). Lungs were examined for histopathological changes at 12, 18, and 24 h p.i. Serial lung sections were stained with hematoxylin and eosin (HE), terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) for detection of apoptotic cells, and an anti-O2 antibody for detection of MT78 and IMT5155. UEL17 and IMT5104 did not cause systemic infections and the extents of lung colonization were two orders of magnitude lower than for the septicemic strains MT78 and IMT5155, yet all four strains caused the same extent of inflammation in the lungs. The inflammation was localized; there were some congested areas next to unaffected areas. Only the inflamed regions became labeled with anti-O2 antibody. TUNEL labeling revealed the presence of apoptotic cells at 12 h p.i in the inflamed regions only, and before any necrotic foci could be seen. The TUNEL-positive cells were very likely dying heterophils, as evidenced by the purulent inflammation. Some of the dying cells observed in avian lungs in situ may also be macrophages, since all four avian E. coli induced caspase 3/7 activation in monolayers of HD11 avian macrophages. In summary, both pathogenic and non-pathogenic fecal strains of avian E. coli produce focal infections in the avian lung, and these are accompanied by inflammation and cell death in the infected areas

Intraspecies Gene Variation within Putative Epitopes of Immunodominant Protein P48 of Mycoplasma agalactiae

P48 protein of Mycoplasma agalactiae is used to diagnose infection and was identified as potential vaccine candidate. According to the genetic nature of mycoplasma and variable sensitivity in P48-based serological diagnosis tests, intra species variation of P48 nucleotide sequence investigated in 13 field isolates of difference province of Iran along with three vaccine strains. Samples were collected from sheep and goat and were cultured in modified PPLO broth.  Two pair of primer employed to confirm genus and species of isolates and a pair of primer has developed to amplify the P48 gene. The sequencing results of PCR products were aligned and analyzed besides published sequences in GenBank. T-Cell and B-Cell epitopes and antigenicity of sequence were computationally predicted. The results have shown P48 nucleotide sequences are 99.9% identical in field isolates and vaccine strain of Iran, but analysis of GenBank published sequences have shown  divergence up to 5.3% at the nucleotide level and up to 4.9% divergence in protein level of P48 sequences of Iran isolates and other available sequences in GenBank. Single nucleotide polymorphism exists in 89 positions and variable amino acid was observed at 25 residues. Phylogenetic analyses have shown that Mycoplasma agalactiae isolates fall into three main groups based on P48 nucleotide sequences. Immunoinformatics analysis of all available P48 nucleotide sequences have revealed that gene variation lead to differences in immunological properties, but  the gene in Iranian isolates are conservative and stable. The sequence variation in epitopes can be underlying source of antigen heterogeneity as a result, affect serological tests accuracy. Due to the high level of divergence in worldwide isolates and high degree of similarity in P48 protein of Iranian isolates, designing recombinant P48 protein based on local pattern can increase the sensitivity and consistency of serological test

Protective effect of thymoquinone, the active constituent of Nigella sativa fixed oil, against ethanol toxicity in rats

Objective(s): Long term consumption of ethanol may induce damage to many organs. Ethanol induces its noxious effects through reactive oxygen species production, and lipid peroxidation and apoptosis induction in different tissues and cell types. Previous experiments have indicated the antioxidant characteristics of thymoquinone, the active constituent of Nigella sativa fixed oil, against biologically dangerous reactive oxygen species. This experiment was planned to evaluate the protective effect of thymoquinone against subchronic ethanol toxicity in rats. Materials and Methods: Experiments were performed on six groups. Each group consisted of six animals, including control group (saline, gavage), ethanol-receiving group (3 g/kg/day, gavage), thymoquinone (2.5, 5, 10 mg/Kg/day, intraperitoneally (IP)) plus ethanol and thymoquinone (10 mg/Kg/day, IP) groups. Treatments were carried out in four weeks. Results: Thymoquinone reduced the ethanol-induced increase in the lipid peroxidation and severity of histopathological alteration in liver and kidney tissues. In addition it improved the levels of proinflammatory cytokines in liver tissue. Furthermore, thymoquinone corrected the liver enzymes level including alanine transaminase, aspartate transaminase and alkaline phosphatase in serum and glutathione content in liver and kidney tissues. Other experiments such as Western blot analysis and quantitative real-time RT-PCR revealed that thymoquinone suppressed the expression of Bax/Bcl-2 ratio (both protein and mRNA level), and caspases activation pursuant to ethanol toxicity. Conclusion: This study indicates that thymoquinone may have preventive effects against ethanol toxicity in the liver and kidney tissue through reduction in lipid peroxidation and inflammation, and also interrupting apoptosis

Detection of Colicin genes by PCR in Escherichia coli isolated from cattle in Shiraz-Iran Tahamtan 1

ABSTRACT A variety of probiotic bacteria have been tested to control animal and foodborne pathogenic bacteria in livestock. The mechanism of inhibition of pathogenic bacteria for several of those probiotic microorganisms is mediated by the production of bacteriocins. Colicins are probably the group of bacteriocins that have been most thoroughly characterized. Colicins are antimicrobial proteins produced by one strains of Escherichia coli to suppress the growth of other strains of E.coli. The present study indicated the preparation of colicin from colicinogenic bacteria. A total of three hundred rectal and rumen swabs isolated from health and diarrheic calves located in Fars province feces. One hundred and fifteen strains were confirmed as E.coli by biochemical test. Polymerase chain reaction was used to determine the following genes encoding colicins. Nearly 100% of isolates were contained at least one gene of colicin. The frequency of several classes of colicin was determined. As a result the most detected gene was Ia Ib and the least detected gene was A.N.S 4 . Colicin should be tested to control animal and foodborne pathogenic bacteria in livestock

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD50 of 4.0 × 105 CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD50 of 1.2 × 1012 CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes