نشریه میکروبیولوژی دامپزشکی
سال دوازدهم شماره 1 (پیاپی 32، بهار و تابستان 1395)

Indiscriminate use of antibiotics to treat bacterial diseases in aquaculture causes drug resistant strains of bacteria and decrease the effectiveness of drugs, in addition, due to the accumulation of antibiotics in fish and fish consumers has led. Therefore, replaced with less harmful substances, such as herbal products is necessary. in this study, antibacterial effects of essential oils of Plant, Eugenia caryophyllata, Cuminum cyminum, Rosemarinus officinalis and Mentha spicata on the Aeromonas hydrophila, Yersinia ruckeri and Streptococcus iniae bacteria is studied. To determine the minimum inhibitory concentration of essential oils, standard microdilution method (Broth Microdilution) was used and the minimum bactericidal concentration of essential oils according to their MIC values were obtained. The results showed that the essential oil of Eugenia caryophyllata compared with other oils, the stronger and has a higher inhibitory power and the minimum inhibitory concentration and minimum bactericidal on all of the bacteria respectively 1.56, 3.12 mg/ml were obtained. Rosemarius officinalis oil has less power compared with other oils and the minimum inhibitory concentration and minimum bactericidal on all of the bacteria studied respectively 6.25, 12.5 mg/ml were obtained. Gram-positive bacterium Streptococcus iniae compared with Gram-negative bacteria Aeromonas hydrophila and Yersinia Ruckeri is more sensitive to essential oils was studied. Aeromonas hydrophila bacteria showed the highest resistance to Mentha spicata oil. Acording to the results of this study, the use of Eugenia caryophyllata oil to control bacterial infections caused by strains studied are suggested.

Newcastle and infectious bronchitis are causes severe economic losses in poultry industry. Control of these two important poultry disease is based on biosecurity procedures and vaccination. The objective of this study was to evaluate potency of live combine Newcastle (La-Sota) infectious bronchitis (H-120) vaccine in commercial broilers, in compare with their monovalent vaccine. Five hundred commercial broiler chickens were divided into four groups including three 150 bird treatment groups and one 50 bird (control) group. Chickens in group A were vaccinated with each of Newcastle La-Sota and infectious bronchitis H-120 vaccine, group B was received live combine Newcastle (La-Sota) infectious bronchitis (H-120) Razi vaccine. Chickens in group D were vaccinated with the commercial combine vaccine. Antibody titers against Newcastle and infectious bronchitis viruses were evaluated by haemagglutination inhibition (HI) and serum neutralization (SN) assay, respectively on collected sera at defined times. Chickens in groups A and B had sufficiently specific antibody levels against both viruses. There were no significant differences between body weight and feed consumption between treatment and control groups. Razi Newcastle (LaSota) Infectious bronchitis (H-120) live combine vaccine induces high levels of specific antibody against both viruses by one-time administration.

Avian infectious bronchitis (IB) is an acute and contagious disease, and can be involved respiratory and urogenital systems. It can be cause high mortality with economic losses in industrial poultry. For IB detection, tracheal and lung tissue samples were collected from 30 broiler chicken farms with respiratory syndrome in Shiraz and transported to laboratory in cold chain. After preparation and inoculation of samples into embryonated SPF eggs, samples checked for avian influenza (AI) and Newcastle disease (ND) by hemagglutination (HA) test. Viral RNA was extracted from allantoic fluid by purification kit and RT-PCR optimized for detection infectious bronchitis virus (IBV) with global primers and used specific primers for detection of massachusetts and B/793 serotypes by nested-PCR. From 30 broiler chicken flocks, 6 flocks were positive for IBV, and 4 flocks were positive for B/793 serotype in nested-PCR. Association to this result, use of vaccine with B/793 serotype is necessary for chickens.

Blood samples were collected randomly from 100 cattle and 100 buffaloes slaughtered at Ahvaz municipal abattoir. The sera were separated after centrifugation 3000 rpm for 10 min.) and stored at -20°C till assayed. For detecting serologic response to H1 1 and H3N2 influenza viruses, all samples were treated with kaolin, then hemagglutination inhibition (HI) test was performed. The results of this study showed that, in cattle 87 percent (males: 83.5% , females: 93.9%) and in buffaloes 75 percent (males: 68.6% , females: 86.1%) were positive for H3N2. In cattle 21 percent (males: 14.9% , females: 33.3%) and in buffaloes 27 percent (males: 21.8% , females: 36.1%) were positive for H1N1, and 21 percent of cattle (males: 14.9% , females: 33.3%) and 27 percent of buffaloes (males: 21.8% , females: 36.1%) were positive for both H1N1 and H3N2 viruses. Totally 13% of cattle and 25% of buffaloes were negative. Our findings showed that H1N1 and H3N2 strains of influenza virus have potential to infect cattle and buffaloes in this region. Therefore, further researches should be focused on clinical feature and economic importance of influenza A infection in cattle and buffaloes.

Q fever is a widespread zoonosis caused by Coxiella burnetii. Domestic animals are the most important reservoirs of C. burnetii, which is exerted in the milk, urine, feces and reproductive discharge of infected animals. Inhalation of bacteria is the main route of animal and human infection. The aim of this study was to survey prevalence of C. burnetii in serum and milk of referred cows to veterinary hospital of Shahid Chamran university and correlation of this organism with host and management determinants. Milk and serum samples from 86 cows were collected and were examined by PCR and ELISA assay, respectively. Prevalence of Q fever in milk and serum samples was 4.65% and 3.49%, respectively. Logistic regression showed that the odds of infection was increased with increase of age (OR: 1.17 and 95% CI: 0.89-1.52, P>0.05) and 3.4% of fluctuation of infection was justified by age. Odds of infection in industrial husbandry than traditional was 2.81(95% CI: 0.53-15.02, P>0.05) and 4.1% of fluctuation of infection was justified by husbandry. 3.8% of fluctuation of infection was justified by fertility status and odds of infection in cows with infertility than healthy was 3.16 (95% CI: 0.35-28.37, P>0.05). The result showed excretion of C. burnetii in milk and the fact that bacteria is in correlation with infertility in cows. Thus efforts must be taken to control and prevent Q- fever.

Chitosan, a natural nontoxic biopolymer derived by deacetylation of chitin, has received considerable attention for its commercial applications in the food industry. In the present study, the ability of chitosan and chitosan nanoparticles to sensitize Escherichia coli O157:H7 to low pH was assessed. To achieve this purpose, cells of E. coli O157:H7 were exposed to pH 4 and 5 adjusted by adding hydrochloric and acetic, acids into TSB, in the presence of 0.0, 0.05, 0.1 and 0.2 % chitosan, for one hour. Chitosan did not show antibacterial activity at pH 5, while 0.2 % nanochitosan made E. coli O157:H7 cells more sensitive to this pH. However, antibacterial activity of both chitosan and nanochitosan werehigher at pH 4, where 0.2 % chitosan and 0.1 and 0.2 % nanochitosan showed significant antibacterial effects on E. coli O157:H7 at this pH. In general, the higher antibacterial activity of nanochitosan than chitosan may be due to the nanoparticle's larger surface area and higher affinity with bacterial cells. Results of the fluorescent staining demonstrated that chitosan and nanochitosan exerted their effect by disrupting the cellular membrane of the cells.

Detection of Cytolethal distending toxin (cdt) genes in Escherichia coli isolates from poultry colibacillosis has been reported from variety of pathogenic Gram negative bacteria such as Escherichia coli, Escherichia albertii, Campylobacter spp and Shigella spp. CDT producing Escherichia coli (CTEC) has been isolated from patients with gastrointestinal, urinary tract infection and sepsis. Up to now the source of human infection remains unknown. Since meat and meat products have been incriminated as the predominant causes of many food borne infections and poultry meat is one of the most important sources of animal proteins in Iran, the purpose of this study was detection of Cytolethal distending toxin gene in Escherichia coli isolated from poultry colibacillosis. 246 Escherichia coli isolated from avian colibacillosis (APEC) and fifty-four fecal E.coli isolates from the feces of apparently healthy birds (AFEC) were investigated for the presence of cdt genes. Isolates collected from veterinary medicine laboratories in Tehran and detection carried out with colony hybridization assay. The results showed only 1.62% of isolates possess cdt gene. This finding indicates that poultry meat probably could not be the main reservoir of CTEC. However expression of CDT from other Gram negative bacteria in poultry meat is not unexpected.

Measuring total microbial count of ice-cream with conventional pour plate count method and comparing the results with standard limits is one of the routine tests in ice-cream manufacturer factories. Achieving the results of total microbial count in minimum time is really important for confidence from the hygienic quality of products. So impedance-splitting method as a new technique for this purpose was considered in order to receiving the results in less time and as soon as possible. The main purpose of this study was to evaluate the correlation between impedance detection time (IDT in hrs.) and total microbial population (log 10 N) of industrial and traditional ice-cream in order to design the predictive microbial model according to impedance technique. During 6 months, 120 samples (60 samples for industrial and 60 samples for traditional ice-cream) were collected from different areas of Ahvaz. Half of samples in both industrial and traditional ice-creams were Vanilla and the other half were cocoa. Samples examined under sterile conditions. The total microbial count by pour plate technique and impedance splitting method were carried out based on the recommendations of Iran's Standard Institute and Industrial Investigation. Then the calibration curves of 2 methods and their equations were obtained by using Excel software. The calibration curves of methods were elaborated for total microbial count and impedance detection time, demonstrating a good correlation between the two methods in industrial ice-cream 96.55% and 95.31% for Vanilla or plain and cocoa ice-creams respectively). According to the calibration curves, the correlation between two methods was 89.07% and 87.37% for plain and cocoa traditional ice-creams respectively. In general the correlation between two methods was 94.61% for industrial and 87.92% for traditional ice-cream. Therefore, impedance measurement which is a more rapid, automated and less laborious method than conventional total microbial count technique could be used like some developed countries as an alternative method for the rapid measuring the total microbial loads in foods instead of conventional methods.

The purpose of this study was molecular identification and colonization of Staphylococcus aureus and Staphylococcus pseudintemedius in apparently healthy dogs referred to Veterinary teaching Hospital in Ahvaz. Nasal swabs from 143 dogs referred to Veterinary Hospital were cultured and identified using routine biochemical and molecular (duplex PCR) methods. Based on biochemical methods, 13 isolates (40/19%) were identified as Staphylococcus aureus but a definite diagnosis of the remaining isolates (54 isolates) was not possible. Thermonuclease gene (nuc), specified for Staphylococcus pseudintermedius and Staphylococcus aureus was amplified by PCR. 13 (40/19%) and 54(59/80%) of coagulasepositive staphylococci, were identified as Staphylococcus aureus and Staphylococcus pseudintermedius respectively by PCR. 7(44/10%) dogs showed mixed infection with Staphylococcus aureus and Staphylococcus pseudintermedius. Considering high prevalence of coagulase-positive staphylococci, especially Staphylococcus pseudintermedius in dogs and potential transmission to humans, health care is necessary by the owners in the face of these animals.

Infectious bursal disease or Gumboro, is a highly contagious viral disease of poultry which causes economic losses in the poultry industry, worldwide. Following infection, the first antibodies are produced against the structural VP3 protein of the Infectious bursal disease virus (IBDV); therefore, VP3 is a suitable antigen for use in ELISA. Given the difficulty of purifying native VP3 from IBDV or virus-infected cells, recombinant VP3 expressed in E. coli can be considered for this purpose. This study was performed with the aim of cloning the VP3 gene of D78 vaccine strain of the virus in pMal-C2X plasmid (a prokaryotic expression plasmid) and the expression and purification of the recombinant protein. Thus, VP3 gene was amplified by RT-PCR and cloned in plasmid pMal-C2X, after digestion with appropriate restriction enzymes. After sequencing to ensure the successful cloning of the gene, the expression of recombinant protein was confirmed by SDS-PAGE. In the following, the recombinant protein was purified using amylose resin chromatography column. Evaluation of the recombinant VP3 protein in immunoblotting showed that the produced protein was antigenically active. Considering the high efficiency of protein production in this system, the expressed protein is a good candidate for the design of ELISA to measure antibody titers against infectious bursal disease virus (IBDV).

Pasteurella multocida is a Gram-negative, nonmotile, penicillin-sensitive coccobacillus belonging to the pasteurellacea family, and can cause a common infection in humans and animals. P.multocida also important factor in diseases such as fowl cholera in domestic and wild birds, atrophic rhinitis in pigs, cattle and sheep pneumonia or respiratory disease, hemorrhagic septicemia in cattle and buffalo and sniffle disease in the rabbit. The bacteria types that cause Fowl cholera in bird species normally belong to serotypes (A: 1, A: 3 or A: 4). In this study, thirty strains of P.multocida isolated from poultry were studied using bacteriological and biochemical tests according to the classical methods. Several factors are known as virulence factors of P.multocida. The P.multocida isolates studied in this study were obtained from the poultry in Iran and were examined for the presence of virulence factor capsule gene, ompH, and adhesion fimbriae containing genes ptfA, pfhA, tadD, hsf-1, fimA and toxA. All of the isolates were confirmed as P.multocida by PM-PCR using species specific primers, KMT1. Molecular capsular typing (CAP-PCR) showed that all of the isolates belonged to type A. Of the 30 examined isolates, all (100%) contained ompH ¡ptfA¡ pfhA¡ hsf- 1 and fimA genes, fifteen isolates (50%) had tadD gene and 21 isolates (70%) had toxA. Analysis of sensitivity patterns to 9 antibiotics showed relative and complete sensitivity in all the tested samples. Two isolates (3.33%) were resistant to antibiotics Flumequin and Nalidixic Acid. Sensitivity to antibiotics Penicillin, Ampicillin, Lincospectin, Florfenicol, Tylosin and Tiamulin was 100% and complete. Sensitivity to antibiotics Flumequin, Enrofloxacine was observed in 96.6% and sensitivity to Nalidixic Acid was observed in 80 . The results of this study showed that the avian P.multocida isolates had the genes for the important diseases caused by these bacteria. It is concluded that P.multocida has the potential for pathogenicity in birds in Iran.

The knowledge of physiology of microorganisms such as bacterial pathogens and their growth behavior under various environmental conditions assist not only the prevention and control of bacterial diseases but also help to recognize the optimum condition for producing of their biological productions e.g vaccines. In this study the effect of temperature and pH was assessed on growth behavior of Streptococcus iniae the causative agent of Streptococcosis in fish. The experiments were runned at temperature 25°C, 30°C , 35°C and pH 5.5 ,7 and 8.5. The obtained linear regression showed that the highest growth rate occurred at 35°C and pH 7 as well as at 30°C and pH 5.5 and 8.5,while the lowest growth rate was obtained at 25°C and pH 5.5. Statisticaly the effect of pH on bacterial growth was more effective than temperature . Significant differences were seen in the growth behaviour of the bacterium at different temperature provided in pH 5.5 (P