Microbiological study of the auditory canal in dairy calves with otitis media.

Otitis media (OM) in calves, is caused by different bacteria. OM treatment requires identification of etiological agents and antibiotic sensitivity testing. The gold standard method of bacteriological study of OM is tympanocentesis, but using this technique in farm condition would be difficult. As a hypothesis, it is possible that bacteriologic examining the auditory canal can help to accelerate the bacteriological investigation of OM. This study was conducted with the aim of comparing the microbiota of the auditory canal in healthy calves and calves with OM. The present research which was a case-control study, mainly compared control group (18 swab samples from healthy and non-infected ear) with two case groups (20 swab samples from the non-affected ear and 32 swab samples from the affected ear in unilateral OM, 11 swab samples from both affected ears in bilateral OM). The results of bacteriological investigations showed three categories of bacteria including: pathogens (Staphylococcus chromogenes, Corynebacterium pilosum, Corynebacterium ovis, Pseudomonas aeruginosa, Pasteurella multocida, Proteus vulgaris, Trueperella pyogenes, Klebsiella, Escherichia coli, Mycoplasma bovis), opportunists (Staphylococcus intermedius, Bacillus licheniformis) and commensals (Staphylococcus epidermidis, Corynebacterium bovis, Corynebacterium renale, Bacillus subtilis, Bacillus cereus). Based on the antibiotic sensitivity test of the isolates, enrofloxacin, florfenicol, and gentamicin were the chosen antibiotics for treatment. All affected animals were treated based on bacteriological results and antibiotic sensitivity tests. All treated animals were fully cured. Based on the results, it seems that in calves with OM, examining the microbiota of the auditory canal can be further studied as an alternative to tympanocentesis in farm conditions.

Molecular and sequencing study and identification of novel SeM-type in beta-hemolytic streptococci involving the upper respiratory tract in Iran.

BACKGROUND: Beta-hemolytic streptococci involving the upper respiratory tract cause strangles and strangles-like diseases in horses and cause severe economic damage to the equestrian club each year. Therefore, careful epidemiological study of these bacteria, evaluation of phylogenetic connections and SeM-typing can be useful to determine the source and epidemiological characteristics of the disease outbreak. Isolates were analyzed using molecular and phylogenetic methods and to determine antibiotic resistance pattern in Iranian isolates. Molecular and phylogenetic methods were used to evaluate Iranian streptococcal isolates, and the similarity of the Iranian SeM-97 sequence with other alleles was assessed using the Neighbor-joining method with the Kimura 2 Parameter statistical model. The amino acid sequence of this gene was compared with the predicted SeM-3 reference amino acid sequence (FM204883) using MEGA 7 software. RESULTS: One type of SeM was found among streptococcal isolates. This type (SeM-97) was reported for the first time and was a new SeM. The relationship between streptococcal isolates and age, sex, race, clinical signs and geographical area was investigated. A significant relationship was observed between streptococcal isolates with age variables and clinical symptoms. CONCLUSIONS: In our study, a Streptococcus equi subsp. equi genotype was identified. The 97 allele of this gene has not been officially reported anywhere and is only registered in the Public databases for molecular typing and microbial genome diversity (PubMLST)-SeM database by Katy Webb. This was the first isolate reported and registered in the mentioned database. The isolate (Tabriz61) had the SeM-97 allele with clinical signs including mucopurulent discharge, abnormal sounds in lung hearing, warmth and enlargement or discharge and abscess of retropharyngeal lymph node and fever. This isolate was sensitive to penicillin, meropenem, ampicillin, cefotaxime, tetracycline, erythromycin, azithromycin, chloramphenicol, enrofloxacin and ciprofloxacin antibiotics and resistant to trimethoprim-sulfamethoxazole and gentamicin antibiotics.

Antimicrobial susceptibility, virulence genes and genomic characterization of Trueperella pyogenes isolated from abscesses in dairy cattle.

Trueperella pyogenes is an opportunistic animal pathogen mainly associated with various suppurative infections in wild and domestic animals. Limited studies have investigated the pathogenesis of diseases caused by this pathogen. The main objective of the current study was to investigate the prevalence, phenotypic properties, virulence genotypes, antimicrobial susceptibility and genetic characterization of T. pyogenes isolated from abscess lesions in different tissues of on-farm dairy cattle. The study was performed on 150 postpartum cattle with clinical abscess symptoms on 22 farms around Tehran, Iran. Classical and disk diffusion methods are used for phenotypic characterization and antibiotic susceptibility. Detection of virulence factor encoding genes and genomic characterization of the isolates also are carried out by conventional PCR and BOX-PCR assays, respectively. Sixty-eight T. pyogenes strains (45.3%) were isolated, 12 were identified as pure cultures and the other 56 strains were isolated from mixed cultures. Seven distinct biotypes were identified among the T. pyogenes isolates. The isolates were mostly resistance to trimethoprim-sulfamethoxazole (70.6%), erythromycin (36.7%), tetracycline (26.5%) and tylosin (23.5%) antibiotics. Also, the genes plo, nanH, nanP and fimA were detected in all isolates. Forty-two isolates (61.7%) carried all virulence factor genes detected in this study. Three isolates only carried plo, nanH, nanP and fimA genes were identified as the least frequent genotype. All sixty-eight isolates and the reference strain were categorized into seven main clusters (A-G). A strong association was observed between virulence factor encoding genes, pathogenicity and biochemical biotypes in some specific clonal relationships.

Assessment of the duration of maternal-derived antibodies specific to the Mycoplasma agalactiae vaccine in goat kids.

BACKGROUND: Contagious agalactia (CA) is one of the most important diseases in the small ruminant industry in Iran. The historical aetiology of this disease is Mycoplasma agalactiae (Ma). The main way to control this disease, in addition to management measures, is vaccination. In ruminant newborns, determining the age of first vaccination against Ma is a challenge due to the interference between colostrum-derived maternal immunity and vaccination-induced immunity. The aim of this study was to evaluate the consistency of maternal-derived antibodies specific to the Ma in goat kids blood serum born from the vaccinated does. OBJECTIVES: Dtermination of level of antibody against Ma in goat kids born from vaccinated dams against Ma. Assessment of duration of protective level of maternal derived antibody in goat kids serum, after receiving colostrum from vaccinted mother with Ma vaccine. Determination the best time vaccination against Ma in goat kids receiving colostrum from vaccinated dams. METHODS: 20 Saanen goat kids were studied in two groups of 10 animals including control (receiving colostrum from unvaccinated does) and treatment (receiving colostrum from vaccinated does). Indirect Elisa was used to evaluate serum specific antibodies to Ma in goat kids (control and treatment groups) from birth to 100 days of age. RESULTS: After receiving a sufficient amount of colostrum, the goat kids in the treatment group had a significantly higher S/P% than the control group until 56 days after birth (p < 0.05) and at 70-100 days after birth, there was no significant difference between the treatment and control groups (p > 0.05). CONCLUSIONS: This study showed that 56-70 days of age could be a good age to give the first dose of CA vaccine in goat kids, but more studies are needed on the effectiveness of this vaccine at this age.

Expression of virulence factor genes in co-infections with Trueperella pyogenes isolates and other bacterial pathogens; an in vivo study.

Trueperella pyogenes is an opportunistic bacterial pathogen causing several infectious diseases, including metritis, mastitis and abscesses in domestic animals such as dairy cattle. Several virulence proteins are released by T. pyogenes strains contributing to the pathogenic and causing disease potential of this pathogen. So far, many aspects of T. pyogenes pathogenesis are unknown. In this study, expression levels of plo, fimA, nanH and cbpA genes encoding pyolysin, fimbriae, neuraminidase and collagen-binding protein, respectively in T. pyogenes isolated from totally 15 metritis, mastitis and cutaneous abscesses convenience samples in response to co-culture with other pathogens including E. coli, St. dysgalactiae, S. aureus, F. necrophorum and L. plantarum strains in mice study model have been investigated. We found that expression levels of plo, fimA, nanH and cbpA genes in T. pyogenes isolates in response to co-culture with F. necrophorum and E. coli were significantly increased; however, no significant changes was seen in the level of expression of these genes in the isolates in response to co-culture with St. dysgalactiae and S. aureus. Notably, expression of all virulence factor genes was suppressed in T. pyogenes in response to co-culture with L. plantarum. We observed that L. plantarum might be used to prevent infectious diseases caused by T. pyogenes.

Cuminum cyminum L. Essential Oil: A Promising Antibacterial and Antivirulence Agent Against Multidrug-Resistant Staphylococcus aureus.

Cuminum cyminum L. (cumin) is valued for its aromatic and medicinal properties. There are several reports of antibacterial activity of C. cyminum essential oil (CcEO). Accordingly, the present study was conducted to investigate the mechanism(s) of action of the CcEO against multidrug-resistant (MDR) Staphylococcus aureus. Therefore, 10 S. aureus MDR isolates, obtained from different sources, were selected based on the antibiotic susceptibility patterns and the Clinical and Laboratory Standards Institute definition and subjected to the examinations. Our results exhibited promising bacteriostatic and bactericidal properties of the CcEO. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration values ranged from 5 to 10 and 10 to 20 µL ⋅ mL-1, respectively. Scanning electron microscope was used to assess the bacterial cell structure and morphology after the induction with 1/2 MIC concentration of the CcEO. The observed morphological changes appeared to be deformation of the cell membrane and destruction of the cells. In the case of quorum sensing inhibitory potential, treatment of S. aureus isolates with the sub-MIC concentrations (1/2 MIC) of the CcEO significantly reduced the hld expression (3.13-fold downregulation), which considerably controls S. aureus quorum-sensing accessory regulator system. Another virulence factor influenced by the CcEO was the polysaccharide intercellular adhesion production system, as an important component of cell-cell adhesion and biofilm formation. Consequently, the expression level of the intercellular adhesion (ica) locus in the S. aureus cells was examined following treatment with CcEO. The results showed significant decrease (-3.3-fold) in ica expression, indicating that the CcEO could potentially interfere with the process of biofilm formation. Using the ethidium bromide efflux inhibition assay, the S. aureus NorA efflux pump was phenotypically but not genotypically (in quantitative polymerase chain reaction assay) affected by the CcEO treatment. Using gas chromatography-mass spectrometry analysis, cuminic aldehyde (38.26%), α,ß-dihydroxyethylbenzene (29.16%), 2-caren-10-al (11.20%), and γ-terpinene (6.49%) were the most detected compounds. The antibacterial and antivirulence action of the CcEO at sub-MIC concentrations means that no microbial resistance will be promoted and developed after the treatment with this agent. These findings revealed that the CcEO is a promising antibacterial agent to control infections caused by the MDR S. aureus strains.

Pathogenic Salmonella weakens avian enriched blood monocytes through ATP depletion, apoptosis induction and phagocytosis inefficiency.

Salmonella enterica Subsp enterica serovar Typhimurium (S. Typhimurium, ST) is one of the most important serovars of the genus Salmonella in human and animals. Because of its intracellular tropism, monocytes/macrophages are pivotal in killing of Salmonella serovars; they are also responsible for transporting of ST to extra-intestinal organs. To investigate the effect of the ST on the functions of avian innate immune cells, almost homogeneous enriched monocytes (EMo) were isolated from peripheral blood mononuclear cells of 2-3 weeks-old of healthy broilers. The EMo were then divided in three groups: control (media only), treatments (challenged with ST clinical isolates) and [doxorubicin (Dox), specifically as positive control for EMo apoptosis] groups. Cellular-molecular damage caused by ST in EMo was assessed with bioluminescence (for caspase-3, 7, and 9 activities and intracellular ATP content), chemiluminescence (for pro/anti-oxidant capacities) and flow cytometry (for apoptosis/necrosis). Further, phagocytosis capacity of post-ST challenged EMo was assessed using a flow cytometry-based internalisation of FITC-loaded polystyrene microparticles. Like the effects of Dox, in post-ST challenged EMo much higher caspase-3, 7 and 9 activities and ATP depletion along with decreased phagocytosis capacity and anti-oxidant load were observed. The results herein indicate that ST weakens EMo particularly through caspases activation/apoptosis. These findings can open a new window on the molecular aspects of Salmonella-macrophage interactions and immunopathology/pathogenicity of salmonellosis in animals especially avian species.

Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran.

Background & objectives: Diarrhoeagenic Escherichia coli strains are common agents of diarrhoea particularly in developing countries. Food products of animal origin are considered as common carriers of E. coli. This study was undertaken to identify enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) pathotypes in animal-source foods (ASF). Methods: A total of 222 ASF samples were investigated. Based on the culture and biochemical tests, 109 E. coli isolates were identified. Duplex-polymerase chain reaction assay was used to detect ETEC and EPEC. The target genes selected for each category were the lt and st for the ETEC, and eae and bfp for the EPEC isolates. Results: The occurrence of E. coli in dairy and meat products was 45 and 52.5 per cent, respectively. Among the E. coli isolates, two ETEC, one typical EPEC and three atypical EPEC were detected in meat samples, whereas only one typical EPEC and one atypical EPEC were detected in dairy samples. Interpretation & conclusions: Our results showed presence of ETEC and EPEC strains in ASFs. The milk without pasteurization and traditional dairy products produced in unhygienic conditions are most likely the main sources of E. coli pathotypes and other zoonotic pathogens and thus can be considered a potential hazard to the health of the community.

Antimicrobial effect of Licochalcone A and Epigallocatechin-3-gallate against Salmonella Typhimurium isolated from poultry flocks.

BACKGROUND AND OBJECTIVES: Salmonellosis due to multi-drug resistant Salmonella Typhimurium with biofilm formation ability is a serious public health threat worldwide. Studies have shown that medicinal plants inhibit the growth of bacterial species. The present study aimed at determining antibiotic resistance pattern and biofilm formation ability of S. Typhimurium isolated from poultry flocks. Moreover, the antibacterial activity of Licochalcone A (LAA) and Epigallocatechin-3-gallate (EGCG) against the studied isolates were investigated in this study. MATERIALS AND METHODS: Antibiotic susceptibility testing of S. Typhimurium RITCC1730 and 23 clinical isolates of S. Typhimurium against 8 antibiotics was performed using standard Kirby-Bauer disc diffusion method. The extent of biofilm formation was measured by Microtiter dish biofilm formation assay. Antimicrobials activities of LAA and EGCG were determined by MIC and MBC assays using microdilution method. RESULTS: The highest antimicrobial resistance was detected against chloramphenicol (52.17%), followed by furazolidone (26.08%), and trimethoprim/sulfamethoxazole (21.73%). All isolates were sensitive to ciprofloxacin (100%), followed by gentamicin, imipenem (95.65%), and cefixime (91.30%). Most of the isolates (78.26%) were able to produce weak biofilm. LAA and EGCG inhibited the growth of S. Typhimurium at the MIC levels of 62.5∼1000 and 1.56∼400 µg/mL, respectively. The MBC value of LAA was >1000 µg/mL, while the corresponding value of EGCG varied from 100 to 800 µg/mL. CONCLUSION: S. Typhimurium isolates revealed a multiple antibiotic resistance with biofilm production ability. As a result, EGCG, and to a lesser extent, LAA displayed potential antibacterial activity against S. Typhimurium and could be considered as useful compounds for the development of antibacterial agents against salmonellosis.

Pathotyping of diarrhoeagenic cattle Escherichia coli strains isolated in the Province of Tehran, Iran.

An oligonucleotide DNA microarray targeting 348 virulence factors and genetic markers was used in the pathotyping, serotyping and phylogrouping of 51 Escherichia coli strains isolated from faecal samples. The samples were collected from diarrhoeic 1 to 30 days old calves located at 14 farms in the Tehran province, Iran. Positive microarray signals for genes encoding the Locus of Enterocyte E acement (LEE), the Type III Secretion System (TTSS), and the absence of EPEC adherence factor (EAF) permitted the pathotyping of 25 strains as atypical Enteropathogenic (aEPEC) or Enterohaemorrhagic Escherichia coli (EHEC). The lack of LEE and TTSS-associated genes distinguished the remaining 26 strains, which were classi ed as Extraintestinal pathogenic E. coli (ExPEC). Atypical EPEC belonged to phylogroup B1 and possessed a LEE pro le tir-1, eae(beta), espA-1, espB-3. The EHEC strains primarily belonged to the B1 phylogroup type-O26 and possessed either a LEE pro le tir-1, eae(beta), espA-1, espB-3, or a B1 type-O111, LEE tir-3, eae(gamma), espA-1, espB-2. ExPEC-typed strains generally harboured genes localised to the constant region of Colicin V plasmid (pColV), including increased serum survival factor (iss), complement resistance protein (traT), aerobactin operon (iucD), and the siderophore receptor (iroN). The microarray platform used in this study is well suited to accurately and rapidly type attaching and e acing E. coli (AEEC-types), thus providing a database for the meta-analysis of ExPEC-typed strains.

Molecular and serological detection of Ehrlichia canis in naturally exposed dogs in Iran: an analysis on associated risk factors.

The general aim of this study, which was conducted for the first time in Iran, was to evaluate the seroprevalence and geographical distribution of Ehrlichia canis in a dog population in Iran, followed by molecular confirmation using PCR and sequencing. Blood samples were collected from 240 dogs in different areas of Alborz and Tehran Provinces and initially analyzed using the immunofluorescent antibody (IFA) test to detect anti-Ehrlichia canis IgG antibodies. Subsequently, nested PCR was performed based on a fragment of the 16S rRNA gene of E. canis on serologically positive samples. The results showed that 40/240 dogs (16.6%) presented anti-Ehrlichia canis IgG antibodies and that nine of the blood samples from the 40 seropositive dogs (22.5%) contained E. canis DNA, which was confirmed by sequencing. The seroprevalence of E. canis tended to be higher in purebred, one to three-year-old male dogs living in the Plain zone, in rural areas; however, this difference was not statistically significant.

Molecular and serological detection of Ehrlichia canis in naturally exposed dogs in Iran: an analysis on associated risk factors / Detecção molecular e sorológica de Ehrlichia canis em cães naturalmente expostos no Irã: uma análise dos fatores de risco associados

The general aim of this study, which was conducted for the first time in Iran, was to evaluate the seroprevalence and geographical distribution of Ehrlichia canis in a dog population in Iran, followed by molecular confirmation using PCR and sequencing. Blood samples were collected from 240 dogs in different areas of Alborz and Tehran Provinces and initially analyzed using the immunofluorescent antibody (IFA) test to detect anti-Ehrlichia canis IgG antibodies. Subsequently, nested PCR was performed based on a fragment of the 16S rRNA gene of E. canis on serologically positive samples. The results showed that 40/240 dogs (16.6%) presented anti-Ehrlichia canis IgG antibodies and that nine of the blood samples from the 40 seropositive dogs (22.5%) contained E. canis DNA, which was confirmed by sequencing. The seroprevalence of E. canis tended to be higher in purebred, one to three-year-old male dogs living in the Plain zone, in rural areas; however, this difference was not statistically significant.

O objetivo geral deste estudo, que foi feito pela primeira vez no Irã, foi avaliar a soroprevalência e distribuição geográfica de Ehrlichia canis em população de cães no Irã, seguida da confirmação molecular por meio de PCR seguida de sequenciamento. Amostras de sangue de 240 cães de diferentes áreas das Províncias de Alborz e Teerã foram coletadas e, inicialmente, analisadas pelo Reação de Imunofluorescência (IFA) para detecção de anticorpos IgG anti-Ehrlichia canis Subsequentemente, uma reação do tipo nested PCR baseada em um fragmento do gene 16S rRNA de E. canis foi realizada nas amostras sorologicamente positivas. Os resultados mostraram que 40/240 cães (16,6%) apresentaram anticorpos IgG anti- Ehrlichia canis e nove (22,5%) das amostras de sangue dos 40 cães soropositivos continham DNA de E. canis, confirmado por sequenciamento. A soroprevalência de E. canis, embora não estatisticamente significativa, mostrou uma tendência em se apresentar maior em cães machos com 1-3 anos, de raça pura, que vivem em zonas planas e áreas rurais.

Repetitive sequences based on genotyping of Candida albicans isolates obtained from Iranian patients with human immunodeficiency virus.

OBJECTIVES: Candidiasis infection caused by Candida albicans has been known as a major problem in patients with immune disorders. The objective of this study was to genotype the C. albicans isolates obtained from oral cavity of patients with positive human immunodeficiency virus (HIV(+)) with or/and without oropharyngeal candidiasis (OPC). MATERIALS AND METHODS: A total of 100 C. albicans isolates from Iranian HIV(+)patients were genotyped using specific PCR primers of the 25S rDNA and RPS genes. RESULTS: The frequencies of genotypes A, B and C which were achieved using 25S rDNA , were 66, 24 and 10 percent, respectively. In addition, genotypes D and E were not found in this study. Each C. albicans genotype was further classified into four subtypes (types 2, 3, 2/3 and 3/4) by PCR amplification targeting RPS sequence. CONCLUSION: In general, genotype A3 constituted the majority of understudy clinical isolates obtained from oral cavity of Iranian HIV(+) patients.

Generation of a Uracil Auxotroph Strain of the Probiotic Yeast Saccharomyces boulardii as a Host for the Recombinant Protein Production.

BACKGROUND: Saccharomyces boulardii (S. boulardii) is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. METHODS: Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA (5-Fluoroorotic acid), uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants. RESULTS: Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. CONCLUSION: A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmacuetics into the intestinal lumen.

Assessment of immunity against avian colibacillosis induced by an aroA mutant containing increased serum survival gene in broilers

Colibacillosis is an important disease in the poultry industry which causes serious economic damages. As it is suggested that vaccination is one of the means to control colibacillosis, we tried to investigate the vaccine potential of a ÃaroA derivative of an O78:K80 avian pathogenic Escherichia coli containing increased serum survival gene. 490 chicks were selected as follows: For assessment of virulence of ÃaroA mutant, 30 chicks were divided into three groups and injected with 0.5ml of PBS or bacterial suspension containing either10(7)colony forming units (CFU) of mutant or parent strains via subcutaneous route. Macroscopic lesions and mortality rate were recorded in different groups during the week after challenge. For assessment of safety and immunogenicity of the ÃaroA mutant, three groups of 20 chicks were vaccinated by aerosol administration of 250 ml of suspension containing 10(8) CFU of mutant strain at days 1 and 14, while the two other groups received PBS or wild type strain. Macroscopic lesions and mortality rate were recorded in different groups until day 21. To determine whether the vaccination is protective against challenges or not, the chickens were vaccinated at days 1 and 14 and challenged intramuscularly with either a homologous or heterologous strains at day 21. Macroscopic lesions and mortality rate were recorded in different groups during the week after challenge. The results revealed that the ÃaroA mutant was slightly virulent, however it was safe and did not cause mortality, lesions or weight loss after vaccination. Antibody responses were similar in the control and mutant groups and vaccination did not induce a significant humoral immunity. The mutant could not protect chickens against both homologous and heterologous challenges. This could be due to several factors such as the high amount of maternal antibodies in the first two weeks of life, and the vaccination procedure.

Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory tract specimens by real time PCR.

Staphylococcus aureus is a significant cause of hospital-acquired pneumonia (HAP), particularly in mechanically ventilated patients. We used the fibronectin-binding protein A gene (fnbA) for the species-specific and quantitative detection of S. aureus directly from lower respiratory tract (LRT) specimens by a Taq Man real time PCR. For this reason, a total of 269 lower respiratory tract (LRT) specimens collected from patients with hospital-acquired pneumonia were assayed. Amplification of fnbA in serial dilutions ranged from 10(9) CFU/ ml to 10(2) CFU/ml. Standard curve of triplicate every dilution had slope 3.34±0.1 and R2>0.99 with SD 0.1. Based on these data, the sensitivity and specificity of the newly developed real time PCR targeting the fnbA gene were both 100%. The Cohen's Kappa test showed the Kappa value of 1.0. The fnbA gene is a potential marker for the species-specific detection of S. aureus and can be used to detect this bacterium in any clinical specimens by real time PCR. Moreover, this method reduces the time needed for quantitative detection of Staphylococcus aureus from LRT specimens to nearly 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

Genetic characterization of Escherichia coli O157:H7 strains isolated from the one-humped camel (Camelus dromedarius) by using microarray DNA technology.

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.

Assessment of immunity against avian colibacillosis induced by an aroA mutant containing increased serum survival gene in broilers.

Colibacillosis is an important disease in the poultry industry which causes serious economic damages. As it is suggested that vaccination is one of the means to control colibacillosis, we tried to investigate the vaccine potential of a ∆aroA derivative of an O78:K80 avian pathogenic Escherichia coli containing increased serum survival gene. 490 chicks were selected as follows: For assessment of virulence of ∆aroA mutant, 30 chicks were divided into three groups and injected with 0.5ml of PBS or bacterial suspension containing either10(7) colony forming units (CFU) of mutant or parent strains via subcutaneous route. Macroscopic lesions and mortality rate were recorded in different groups during the week after challenge. For assessment of safety and immunogenicity of the ∆aroA mutant, three groups of 20 chicks were vaccinated by aerosol administration of 250 ml of suspension containing 10(8) CFU of mutant strain at days 1 and 14, while the two other groups received PBS or wild type strain. Macroscopic lesions and mortality rate were recorded in different groups until day 21. To determine whether the vaccination is protective against challenges or not, the chickens were vaccinated at days 1 and 14 and challenged intramuscularly with either a homologous or heterologous strains at day 21. Macroscopic lesions and mortality rate were recorded in different groups during the week after challenge. The results revealed that the ∆aroA mutant was slightly virulent, however it was safe and did not cause mortality, lesions or weight loss after vaccination. Antibody responses were similar in the control and mutant groups and vaccination did not induce a significant humoral immunity. The mutant could not protect chickens against both homologous and heterologous challenges. This could be due to several factors such as the high amount of maternal antibodies in the first two weeks of life, and the vaccination procedure.

Molecular genetic differentiation of avian Escherichia coli by RAPD-PCR / Diferenciação molecular de Escherichia coli aviária por RAPD-PCR

Escherichia coli is one of the most important bacterial avian pathogens and a common inhabitant of the gastrointestinal tract of animals. Most pathogenic E. coli can not be differentiated biochemically or by classic microbiologic methods. Molecular typing methods, particularly PCR, facilitated epidemiological and ecological studies of bacteria. Here we describe the application of a random amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR) for molecular genetic differentiation of E. coli isolates in Iran. In this study 58 E. coli isolates including 4 standard strains, 3 food originated isolates, 33 avian isolates, 8 isolates form diarrheic calves and 10 isolates from unweaned diarrheic lambs were analyzed by RAPD-PCR using primer 1247(5/-AAG AGC CCG T-3/). The RAPD analysis showed that these isolates could be grouped into 33 RAPD types and avian isolates were discriminated into 29 genotypes. It was shown that the primer could not differentiate E. coli isolated from lambs. Discriminatory index for entire isolates was 0.912 and for avian isolates was 0.990. We concluded that RAPD-PCR can be used as a method for molecular differentiation of E. coli isolates.

Escherichia coli é um dos patógenos aviários mais importantes e um habitante comum do trato gastrointestinal de animais. A maioria das cepas patogênicas não pode ser diferenciada por métodos bioquímicos ou outros métodos microbiológicos clássicos. Métodos de tipagem molecular, particularmente PCR, têm facilitado os estudos epidemiológicos e ecológicos a respeito desse microrganismo. Nesse estudo, descrevemos a aplicação do RAPD-PCR para a diferenciação molecular de isolados de E.coli do Irã. No estudo, 58 isolados, incluindo 4 isolados padrão, 3 isolados de alimentos, 33 isolados de aves, 8 isolados de bezerros diarréicos e 10 isolados de carneiros diarréicos foram analisados por RAPD-PCR com o primer 1247 (5'-AAG AGC CCG T-3'). A análise mostrou que esses isolados podiam ser agrupados em 33 tipos RAPD, sendo os isolados de aves agrupados em 29 genótipos diferentes. Verificou-se que o primer utilizado não diferenciou os isolados de carneiros. O índice discriminatório para todos os isolados foi 0,912 e para os isolados de aves foi 0,990. Concluiu-se que o RAPD-PCR pode ser usado como método para diferenciação molecular de isolados de E. coli.

Evaluation of random amplified polymorphic DNA analysis and antibiotic susceptibility application in discrimination of salmonella typhimurium isolates in Iran.

The purpose of this study was to determine the utility of RAPD-PCR and antibiotic susceptibility tests in differentiation of S. typhimurium isolates in Iran. Thirty isolates of S. typhimurium, selected based on animal source; place and time of the isolation and a reference strain of the bacterium were used in this study. Serotyping of the isolates was performed by reliable antisera and confirmed with a multiplex PCR. Genomic DNAs were extracted and subjected to an optimized RAPD-PCR, using two previously reported arbitrary primers (P1254 and 23L). Bacteria were also examined for resistance against 8 antibiotics, using a standard antibiotic susceptibility test. While the antibiotic susceptibility test resulted in the identification of 13 profiles of R-type among the bacterial isolates, application of primers P1254 and 23L in the RAPD-PCR could discriminate the isolates only in four and six profiles respectively. However, combination of the two methods could differentiate the 30 isolates in 20 different profiles. The results of this study indicate that the discriminatory power of RAPD-PCR for S. typhimurium is low but a combination of this method with antibiotic susceptibility test could be considered an easy and relatively reliable discriminatory approach in differentiation of S. typhimurium for epidemiologic purposes in Iran.