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Additional file 1: Fig. 1S. PDHB-P80 sequence. From nucleotide 1 to 1015 is PDHB sequence and 1027 tothe end is P80 antigenic region. Fig. 2S. The hydrophobicity of whole P80 sequence estimated by BioEdit Sequence Alignment Editor. The more antigenic (hydrophilic) regions of P80(from amino acid 373 to 524) were selected to construct the fusion protein. Fig. 3S. Separated solubility analysis of PDHB and P80 using Pro-Sol online server. Fig. 4S. Secondary structure of PDHB protein estimated by SOPMA online server. Fig. 5S. Secondary structure of P80 protein estimated by SOPMA online server.

Generally, the high widespread presence of antimicrobial resistance, and the next freeing into aquatic environments which provide a situation for transmission of these genes in water is because of the abuse of the antimicrobial drugs in both medicine and veterinary medicine. In aquatic environment, bacteriophages could have an important role in sharing antimicrobial resistance genes. The purpose of this study was to assess three important antibiotic resistance genes including two β-lactamases (blaTEM, blaSHV) and sul1 gene, referring to resistance to sulfonamides, in both bacteria and phage DNA fractions of wastewater samples, Shiraz, Iran, using polymerase chain reaction. The prevalence of those genes was extremely high and equal to 100% in bacterial DNA, while these rates were lower in phage DNA fractions as 66.66%, 66.66% and 58.33% for blaTEM, blaSHV and sul1, respectively. In conclusion, detection of mentioned genes in bacterial and phage DNA fractions from ambient water is considerable, so the possibility of harbouring and transferring of antibiotic resistance genes by phages needs to be explored in the future. Also, available data is a reputable endorsement that wastewater is a hotspot for these kinds of genes to spread in the environment. Based on our knowledge, this is the first report of blaTEM and bla SHV and sul1 genes in bacterial and phage DNA fractions insulated from urban wastewater and environment in Iran.

The microbial population of the uterus fluctuates during the estrous cycle. Microflora of uterus may affect the establishment and maintenance of pregnancy in bitches. The endoscopic samples obtained from the vagina and uterus of 20 female adult mixedbreed dogs. The uterine lavage samples were prepared for cytology, bacterial (aerobic and anaerobic) and fungal cultures. Uterine tissue samples were evaluated for the presence of E. coli by the polymerase chain reaction. The pure growth of bacteria was observed in seven plates out of the nineteen cultured samples (36.84%) and five Gram-negative and two Gram-positive bacteria were detected. The highest number of isolated bacteria was related to the samples of the diestrus and anestrus stages of the estrous cycle, while the lowest number of bacteria was observed in the samples of the estrous stage. Moreover, Citrobacter spp. was the most frequent group of isolated bacteria. The neutrophils were detected in the cytology of uterine samples. The fungi growth was observed in three uterine samples. Cladosporium and Penicillium isolated from the samples were related to the estrus stage, and yeast was grown in diestrus samples. The 16srRNA gene existed in all of the estrous uterine samples in which the bacterial culture was negative. However, the presence of this gene was proven in two samples (33.30%) of negative bacterial culture samples from the diestrus and anestrus stages. In conclusion, the normal bitches’ uteri were infected with various bacteria in estrus, diestrus and anestrus stages of the estrous cycle, and it could coincide with the fungi infection.

Additional file 1: Supplementary Table 1. Posterior medians and 95% probability intervals (PrIs) for the Sensitivity (Se) and the Specificity (Sp) of IP-ELISA, Mt-ELISA, f-PCR, Culture.

The aim of this study was to construct, expression of a novel recombinant chimeric protein consisting of Pyruvate dehydrogenase beta subunit (PDHB) and high antigenic region of integral membrane lipoprotein P80 of Mycoplasma agalactiae as a potential diagnostic tool. The full-length sequence of pdhb and a portion of antigenic regions of P80 were selected and analyzed by CLC main workbench 5.5 software. Several linkers and three dimensional structure of PDHB-P80 were compared to the native PDHB and analyzed to select a proper one for expression. The fusion gene sequence was optimized and synthesized in pMAT cloning vector. The synthetic pMAT-pdhb-p80 was digested using Bam HI and Sal I restriction enzymes and ligated into pMAL-p5X expression vector. The pMAL-pdhb-p80 construct was transfected into E.coli BL21 strain cells and expressed protein were purified using amylose resin. and the purified protein was analyzed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In silico analysis demonstrated that fusion proteins using IgG4 middle hinge (CPSCP) with TM-score of 0.99 showed the higher similarity between three dimensional structure of PDHB before and after fusion with high antigenic region of P80. Successful cloning verified by PCR colony, double digestion and sequence analysis. Besides, SDS-PAGE analysis and Western blotting indicated and confirmed the expression of intact recombinant chimeric protein MBP-PDHB-P80 along with some truncated forms of the recombinant protein. it could be concluded that the fusion construct has a potential for serodiagnostic assay in future studies.

Additional file 1: Fig. S1. The uncropped images of Fig. 4. Fig. S2. The uncropped images of Fig. 5. Fig. S3. The uncropped images of Fig. 6.

Zahra Naziri, Abdollah Derakhshandeh, Arash Soltani Borchaloee, Meisam Poormaleknia, Negar Azimzadeh Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, IranCorrespondence: Zahra NaziriDepartment of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz 71345– 1731 Tel +98 71 36138668Fax +98 71 32286940Email z.naziri@shirazu.ac.irBackground: Global increase in the prevalence of virulent extended-spectrum beta-lactamase (ESBL)-producing uropathogenic Escherichia coli (UPEC), which is also multi-drug resistant (MDR), leads to increase in severity of urinary tract infections (UTIs), decrease in the efficacy of the first-line antibiotics, and therefore increase in the morbidity and mortality rates.Methods: We investigated the distribution of ESBL-producing UPEC in 78 E. coli isolates from community-acquired UTI patients in southern Iran. The prevalence of three major ESBL genes, antimicrobial resistance patterns against 15 conventional antibiotic disks, and the presence of 11 important virulence genes that involve in the development and progression of UTIs were evaluated in these isolates.Results: Of the UPECs, 34.6% were ESBL-positive and 96.3% of the ESBL-producers were MDR. Among the ESBL-producers, 100% harbored blaCTX-M, 63% harbored blaSHV, and 11.1% harbored blaTEM genes. ESBL-producers showed a higher level of resistance to the tested cephalosporins, fluoroquinolones, trimethoprim-sulfamethoxazole, and tetracycline than non-ESBL producers. All isolates were resistant to the tested penicillins. Prevalence of resistance to about two-third of the tested antibiotics was higher than 50% and 93.6% of the isolates were MDR. High prevalence of virulence factors particularly the adhesins (82.1% csgA, 73.1% fimH genes) and siderophore (73.1% sitA gene) was seen in the UPECs. But fortunately in MDR isolates, the virulence score and prevalence of hemagglutinin (tsh), hemolysin toxin (hlyD) and invasin (ibeA) genes were lower than in non-MDR UPECs. Shockingly, among the 15 common antibiotics, only nitrofurantoin (< 20% resistance) could be recommended as an appropriate drug for the treatment of UTIs due to our ESBL-producer UPECs.Conclusion: The alarming level of virulent MDR ESBL-producer E. coli strains in this study necessitates the performing of an antibiotic stewardship program, regional screening of ESBL-producers and their virulence properties to select appropriate antibiotic, or designing new therapeutic methods for UTIs by inactivation of the essential virulence factors of UPECs.Keywords: uropathogenic Escherichia coli, extended spectrum beta-lactamases, multi-drug resistance, virulence factor, urinary tract infection

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in ruminants and there has been a shift in the public health approach to MAP and human diseases like Crohn's disease. The prevention of infection by MAP in ruminants is thought to deter the high impact of economic losses in the level of dairy industry and possible spreading of this pathogen in dairy products. The present study was done to investigate the construction and expression of the soluble form of a novel fusion protein, consisting of Heparin-binding hemagglutinin (HBHA) and high antigenic region of Fibronectin Attachment Protein-P (FAP-P), in order to introduce as a Th1 inducer subunit vaccine against MAP. HBHA is a mycobacterial adhesin and it has been demonstrated that a HBHA-specific IFN-γ response, in latent M. tuberculosis infection, depends on the methylation of the antigen. Further, FAP-P induces Th1 polarization. Because methylation of HBHA was not performed in E. coli, Pichia pastoris was chosen as the host. The desired fusion protein had a similar 3D structure to that of HBHA with its native form and post-translational methylation in C-terminal. Hence, the uptake of the purified fusion protein will be done by M cells because of HBHA, and cell-mediated immunity will be induced because of both antigens. Eventually, successful construction and expression of the newly-designed chimeric protein under the mentioned conditions is reported in this article.

Paratuberculosis (John's disease) is infectious and chronically progressive granulomatous disease which affects domestic and wild ruminants. The causative agent is Mycobacterium avium paratuberculosis (MAP), a slow growing mycobactin dependent acid-fast bacillus. We investigated the detection and frequency of MAP in apparently healthy dromedary and Bactrian camels by insertion sequence 900 (IS900) polymerase chain reaction (PCR) and acid fast staining of fecal samples in Iran. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. Although the percentage of positivity for PCR assay of fecal dromedary camel was 8.0%, no bands corresponding to MAP detect in all samples of Bactrian camels. In conclusion, Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cow, sheep and goat.