نشریه میکروبیولوژی دامپزشکی
سال دهم شماره 2 (پیاپی 29، پاییز و زمستان 1393)

Avian infectious bronchitis (IB) is caused by corona viruses. Infectious bronchitis virus (IBV) is a major cause of economic losses in poultry and can be involved in respiratory disease، nephritis، and both poor egg production and quality by reproductive tract infection. IBV has many serotypes that do not confer cross protection against each other. The study in 2012 was conducted to detect IBV in broilers chicken farms suspected of respiratory syndrome with a high mortality and no history of vaccination against of the IBV in Charmahal va Bakhtiyari province. In this study، one hundred serum samples and one hundred tracheal and lung tissue samples were collected from 10 broiler chicken farms with respiratory signs. To achieve this goal، two tests have been used throughout the studies of protocol Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The presence of IBV antibodies was detected by using ELISA kit. The results indicated that 7 flocks (70%) were positive and 3 flocks (30%) were negative tested with RT- PCR method. The evaluation of serum antibody titers of flocks showed that antibodies against infectious bronchitis virus in RT-PCR positive flocks were higher than RT-PCR negative flocks. The results also evinced that the prevalence of infection bronchitis in broiler flocks was high in broilers farms of Chaharmahal va bakhtiyari province and concurrent Infection of IBV and other respiratory pathogen played a role in high mortality rate of respiratory disease.

Neospora caninum is one of the cattle abortion causes throughout the world. It has a significant economic impact on cattle production. The objective of the present study was to determine the infection rate of cattle to N. caninum and defining its related risk factors in dairy cattle farms of Sanandaj، west Iran. A total of 336 sera collected from dairy cattle farms in Sanadaj nd examined by commercial N. caninum IDEXX Elisa kit. N. caninum antibodies were detected in 64 (17. 6%) of the cattle. The infection rates of this protozoan parasite in cattle، reared in industrial and native farms، were 9. 03% and 25. 8%، respectively (P<0. 001). According to the age، it was observed that the infection rate increases upon the older ages.. The infection to N. caninum in lactating cattle was 22. 89%، whereas in non-lactating cattle was 10. 06%. This difference was significantly revealing (P=0. 002). Among the farms with dogs، the percentage of seropositive cattle was 46. 8%، whereas in farms without dogs it was 7. 43%. The difference was highly significant (P<0. 001). This finding seems to be a good indication of dog influences on transmitting the parasite to cattle. In addition، the infection rate in cattle with the history of abortion was significantly higher than the rate of infection in cattle without the history of abortion (P<0. 01). However، there were no significant differences among the infection rates of the pregnant and non-pregnant cattle (P = 0. 669). This project was performed، for the first time، in Sanandaj and the Kordestan province.

Pseudomonas spp. are zoonotic bacteria associated with human and fish accompanied to hemorrhage in the fish. Rapid detection of this disease leads to in time treatment. Present study was performed aiming to compare two kits including API 20 NE and API 20 E with routine biochemical tests to detect pseudomonas species. With occurrence of serious losses in the reared Hypophthamichthys molitrix in Guilan province during spring and summer when environmental conditions were suitable for bacterial infection outbreak، 126 fish from 25 warm water farms locating in Rasht were transferred to the fish health and disease department of Inland water aquaculture institute. After carefully dissection، Samples of kidney، liver، spleen and ascetic fluid of each fish were aseptically streaked onto blood Agarthen incubated for 24-48 hours at 30 ºC. Simultaneously، routine biochemical techniques and API kits were used for characterization. Antibiotic resistance pattern for all pseudomonas isolates was determined using disk diffusion technique on Moller- Hinton agar according to Bauer، et al method (1966) modified based on NCCLS (2007). Based on the results of 75 isolates، 9 isolates (12%) were pseudomonas spp.. Nine isolates were placed in 2 species of P. aeruginisa and P. fluoresences using API 20NE with 98% accuracy While API 20E detected P. aeruginisa with 64% accuracy and P. fluoresences was defined as P. fluoresences/putida. In the antibiotic sensitivity test، gentamycin was the choice drug for both species and for P. fluoresences، Kanamycin and neomycin also had 100% influences. Results indicated that among 2 API systems، API 20NE strip is capable to detect pseudomonas species in fish with higher precision and dissociation ability compared to API 20E.

The American foulbrood (AFB) and European foulbrood (EFB) are important bacterial diseases in honeybee caused by Paenibacillus larvae and Mellisocuccos plutonius، respectively. The aim of this study is to compare the conventional method with molecular method (PCR) in terms of identification of AFB and EFB. In the present study، 30 larves with clinical symptoms from 30 apiaries located in different cities of Lorestan province were collected. The samples were examined for the disease using conventional methods and PCR simultaneously. Two specific primers for 16S rRNA gene were used in PCR. The number of positive samples was 13. 3% in PCR but 10% in the conventional method such as bacterial culture for Paenibacillus larvae. All samples in both bacterial culture and PCR methods were negative for Mellisocuccos plutonius but in the bacterial culture، they were isolated from 6 samples of Paenibacillus alvei، an indicator of European foulbrood. According to the results، PCR is a specific method for the identification of Paenibacillus larvae، so this method can improve the diagnosis tests and be an alternative for the conventional culture methods in the diagnosis of Paenibacillus larvae. But for the Mellisocuccos plutonius، further research to improve the diagnosis tests is suggested.

The clinical signs of heat intolerance syndrome were observed in 70 Holstein cows in a private farm in Isfahan two months after recovery from foot and mouth disease consequences. The condition was characterized clinically by intolerance to increased environmental temperatures. The other signs were panting، hirsutism، profuse salivation and significantly reduced milk production. Blood samples were collected from 10 diseased cows with FMD record، 10 apparently healthy cows with FMD record and 10 healthy cows without FMD record. One cow with the sign of severe panting was slaughtered and the samples from her thyroid، adrenal glands and pancreas were taken for the possible pathologic changes. There were significant increase in blood glucose and phosphorus with a decrease in T3 concentration and neutrophils count in cows with heat intolerance. Pathological changes were necrosis in the thyroid، adrenal glands and pancreas cells. This seems to be the first report in Iran to describe the clinical، pathological and haematobiochemical changes in heat intolerance syndrome in cattle following foot and mouth disease.

Leptospirosis is one of the most important zoonosis with worldwide distribution. lipL41 is an immunogenic outer membrane protein found in pathogenic Leptospira species and may be used in diagnostic method and also can be a good candidate for recombinant vaccine against leptospirosis. The aim of this study was designing a positive control for molecular diagnosis of pathogenic Leptospira spp. by PCR based on lipL41 gene. Five pathogenic serovars and one saprophytic species were used in this study. The serovars were subcultured into the selective culture medium (EMJH) and then the genomic DNA extracted. The lipL41 gene amplified using the specific primers. The PCR product were ligated in pTZ57R/T vector and transformed in competent E. coli Top10 cells. The confirmation of the recombinants was made by picking the white colonies and carrying out colony PCR amplification of the gene. The recombinant plasmids were extracted using a commercial extraction kit. PCR amplification of the lipL41 gene using the specific primers resulted in a 1065 bp in all five pathogenic serovars tested. No PCR products were amplified from the non-pathogenic L. biflexa. Positive colonies plasmid vector was isolated from cells by kit. PCR test was carried out with positive control and PCR products were observed on gel electrophoresis. Due to slow growth of Leptospira، and because of limited diagnostic capacity using a rapid and accurate molecular tests like PCR with a positive control to confirm its accuracy is essential. Therefore، the cloned lipL41 gene in this study that has been prepared in Leptospira reference laboratory can be used as a positive control in all laboratories using the PCR test.

Infections caused by Pseudomonas aeruginosa have been reported from many hospitals، and according to its opportunistic nature، Pseudomonas aeruginosa invades to immunocompromised patients and controlling the following infection is so hard. Antibiotics misusage beside complex mutation mechanisms in pseudomonas genus led to resistance against many antibiotics. During the past decade، tracing for natural antimicrobial peptide is more considered. Among them، melittin has been extracted from honey bee venom and its antibacterial activity is being examined. The main goal of this study was to evaluate antibacterial activity of melittin against clinical isolates of Pseudomonas aeruginosa. 48 Clinical strains were present from previous study. Honey bee venom was prepared using electrical stimulation and the quality of venom was confirmed by SDS-PAGE. Melittin was isolated from the venom using a linear gradient of acetonitrile and C18 column by Reverse Phase-High Performance Chromatography (RP-HPLC) Technique. Minimal Inhibition and Bactericidal concentration of melittin were examined on clinical isolates of Pseudomonas aeruginosa. Honey bee venom consisted of twenty distinct fractions in which melittin was the major one. MIC50 for melittin against all; mucoid، and non-mucoid sensitive isolates، were 12. 5، 6. 25، and 12. 5 μg respectively. Among mucoid and non-mucoid strains، 94. 73 and 86. 2 % were sensitive to melittin respectively. MBC50 for melittin against total، mucoid and non-mucoid isolates were 50، 37. 5، and 50 μg respectively. Totally، 89. 58 % were sensitive to melittin and 10. 41 % were not inhibited. The results obtained from MIC and MBC tests showed that melittin had significant inhibitory and bactericidal effect on clinical isolates of pseudomonas aeruginosa. According to the results، the less amount of melittin can suppress the growth of mucoid strain of pseudomonas. These strains due to existence of polysaccharide layers around bacteria have less permeability to antibiotics and expose more resistance. This study would be of great value to the treatment of cystic fibrosis patients with pseudomonas infections.

All organisms have nuclease. Deoxyribonucleases (DNase) are from enzyme group that are able to hydrolase phosphodiester bounds of DNA molecule bonds. Staphylococcus pasteuri identified by 16S rRNA marker and biochemical tests، and submitted to GenBank/EMBL with KC170006 accession number. The activity of DNase enzyme was detected through three spectrophotometric، detection of DNase activity on circular and double strand DNA molecule on agarose gel methods. The effects of pH، NaCl and cations like Mg2+، Ca2+،Mn2+andMg2+-Ca2+ treatments were considered. S. pasteuri DNase was able to cut single strand of circular DNA. S. pasteuri DNase was able to cut single strand DNA. Studies on circular DNA showed that the enzyme may nick specifically، whereas pET-21a (+) was restricted to one point. The DNase does not cut double strand DNA. This is the first report about S. pasteuri DNase activity. The most important activity of enzyme was observed in the presence of 0. 6 M NaCl، 1mM Mg2+-Ca2+ and pH 5. 5.