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

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

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 B subunits of cholera and Escherichia coli heat-labile toxins enhance the immune responses in mice orally immunised with a recombinant live P-fimbrial vaccine for avian pathogenic E. coli

This study aimed to investigate the adjuvant effect of recombinant attenuated Salmonella expressing cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) for the P-fimbriae subunit-based vaccine of avian pathogenic E. coli (APEC) in a murine model. The PapA-specific sIgA and IgG responses were significantly enhanced after immunisation with the Salmonella-PapA vaccine in the presence of CTB or LTB. The group immunised with the Salmonella-LTB strain promoted Th1-type immunity, whereas that immunised with the Salmonella-CTB strain produced Th2-type immunity. We concluded that both Salmonella-CTB and -LTB strains can enhance the immune response to PapA, and that the LTB strain may be a more effective adjuvant for APEC vaccination, which requires higher Th1-type immunity for protection. Thus, our findings provide evidence that immunisation with an adjuvant, LTB, is one of the strategies of developing effective vaccines against P-fimbriated APEC

Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens

Detection of Mycoplasma gallisepticum by 16S rRNA PCR with specific primers in clinical samples

Mycoplasma gallisepticum (MG) as one the major pathogens of birds, causes significant economic losses in poultry industry. The main purpose of the present study was to detect Mycoplasma gallisepticum in clinical samples using the 16S rRNA PCR method. For serological screaming test, 18 commercial laying farms and 8 broiler breeder farms were selected and rapid serum agglutination test (RSAT) was performed. For polymerase chain reaction sampling, 10 of the 17 farms that were positive in RSAT were selected and 109 sterile swab samples were collected from the palatine cleft, trachea, air sacs and lungs in each farm. Three swabs from three birds were placed in test tube containing 1 ml of phosphate buffered saline and transferred to laboratory form PCR testing. In this study, specific primers for 16S rRNA gene were used. The aforementioned primers are totally specific for MG and can be differentiated from other Mycoplasmaand bacteria present in the trachea of poultry of the 26 farms examined, 17 farms were positive in RSAT serologic test. The 530 bp PCR product produced by specific primers of all field strains appeared on electrophoresis gel in 46 samples from 10 farms accounting to 42.2%.The 16S rRNA PCR with very high sensitivity can be employed in definitive diagnosis of Mycoplasma gallisepticum infection in vitro

Detection of Mycoplasma synoviae infection in broiler breeder farms of Tehran province using PCR and culture methods

Mycoplasma synoviae (MS) is an important avian pathogen that can cause both respiratory disease and joint inflammation synovitis in poultry, inducing economic losses to the Iranian chicken industry especially breeder farms. The aim of this study was to use the MS specific PCR and culture methods in order to detect of M. synoviae from breeder farms where located in Tehran province. A total of 475 samples including choanal cleft, trachea, ovary and /or joint cavities from 23 broiler breeder farms of Tehran area were collected. Samples were cultured in PPLO broth media supplemented for MS isolation. The bacteria DNAs were extracted by phenol/chloroform method. Specific published primers amplify a 207 bp region of the 16S rRNA gene of MS were used for PCR method. Out of 475 samples, 146 cultures were shown positive and typical Mycoplasma colonies, 85 samples were also identified MS based on agglutination test with specific MS antiserum and the PCR method. A total of 122 samples, a band with 207 bp was shown as MS specific PCR product in electrophoresis. In addition to these 85 samples that were positives in both culture and PCR, 37 samples that had not grown in Mycoplasma media were positive in MS specific PCR. A total of 292 samples were negatives in both culture and PCR methods. 122 positive samples out of 475 samples (25.7%) were belonged to 7 breeder farms (30.4%). On conclusions, the MS infection of broiler breeder farms of Tehran area was confirmed truly. From the results, as the PCR method reduces the time consuming, an effectiveness and efficient for detection of M. synoviae infection of chicken breeder. It is then suggested that the PCR method could be an alternative method for culturing

Sequences of mycoplasma hominis in patients with urinary tract infection in a hospital in Kashan, Iran

Mycoplasma hominis is normally found in the urinary tract of human and its role in Urinary Tract Infection (UTI) has been proved. This bacterium causes inflammatory responses and accumulation of leucocytes in urethra. In spite of the presence of the bacteria, the urine culture might be negative. Mycoplasma hominis can be transferred sexually and causes human infertility. The present study was conducted to detect and identify Mycoplasma hominis by molecular methods in urine samples of the patients with UTI, who were referred to our hospital. A total number of 864 urine samples from the patients with UTI were subjected to this study. After routine culture, urine analysis were performed on the samples. The DNA was extracted from the sediments of the urine samples, using phenol and chloroform method. Polymerase Chain Reaction (PCR) was conducted on the extracted DNA to detect the 16S rRNA of Mycoplasma hominis, with the primers; RNAH1 and RNAH2. Based on the results of PCR tests, out of 100 pyuria positive samples, 9 and 1 were infected with Mycoplasma sp. and Mycoplasma hominis, respectively. The sequencing of amplified product of 16S-rRNA revealed a single nucleotide substitution (269 T A), compared with the reference gene of this species. © 2015 Academic Journals Inc

Detection of Mycoplasma synoviae in clinical samples by VlhA-PCR method

As one of the major pathogens of avian species, Mycoplasma Synoviae causes significant economic losses to the poultry industry. The main purpose of this study was to detect Mycoplasma Synoviae in clinical samples using the VlhA-PCR method. For serological screening test, 373 serum samples were collected from 25 breeder farms and rapid serum agglutination test conducted which revealed that 143 samples equivalent to 19 breeder farms were positive. For VlhA-PCR assay, 20 of the previously mentioned breeder farms were selected and sterile swab were collected from the palatine cleft, trachea, air sacs and lungs. Three swabs from 3 birds were placed in a test tube containing 1 ml of PBS and transferred to the laboratory for PCR test. Specific primers for VIhA gene were employed in this study. The PCR product from specific primers showed 350-400 bp for all field isolated on electrophoresis gel in 8 farms. VlhA-PCR with high sensitivity could be employed in definitive diagnosis of Mycoplasma Synoviae infection in the laboratory