فهرست مطالب s. a .pourbakhsh

Mycoplasma gallisepticum (MG) is a contagious avian pathogen that causes financial losses to the poultry industry. Isolation of the pathogen is difficult and time-consuming, and therefore, far from a routine method. Serological testing methods to detect antibodies resistant to MG are widely used in routine diagnosis. Tylosin is a class of macrolide antibiotics tremendously administered in veterinary medicine for the treatment of mycoplasmosis and prophylaxis. This study aimed to detect MG by immunoassay testing, culture, and polymerase chain reaction (PCR) in commercial poultry farms and to investigate the tylosin susceptibility of the isolates. To verify the presence of antibodies resistant to MG, 750 blood samples were randomly collected from 38 broiler farms from 2019 to 2022 in Mazandaran and Golestan provinces, Iran, and rapid slide agglutination (RSA) assay was performed. Positive results were analyzed by the enzyme-linked immunosorbent assay (ELISA) for further investigation. Here, 920 swab samples were collected from 38 non-vaccinated commercial farms for culture, and PCR tests were performed for the isolated strains. The activities of tylosin were tested in vitro against these isolates using the broth microdilution method. The lowest antibiotic concentration that resulted in a color change was considered the minimum inhibitory concentration (MIC) value. Twenty-four (63.1%) farms were positive in the RSA test, and 21 (55.2%) farms were positive in the ELISA test. Nine (23.68%) of the farms grew on culture media, and 8 (21.05%) were detected as Gallisepticum species by PCR. The geometric mean of MIC for tylosin was 5.75 µg/ml, MIC50 was 4 µg/ml, and MIC90 was 8 µg/ml. The results indicated that commercial farms were infected with MG. Considering the ability of MG to spread and the probable use of the RSA test as a rapid and cheap method, it can be argued that ELISA and RSA serological tests can be used to find MG in poultry flocks, and the positive result should be confirmed by standard microbiological tests or PCR. It was also found that the isolated parts of MG changed their sensitivity to tylosin, indicating the need for routine testing to optimize treatment dose and efficiency.

Burkholderia mallei is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of Borkolderia mallei and Burkholderia pseudomallei using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as Burkholderia mallei. The PCR-RFLP assay demonstrated a product for Burkholderia mallei with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for Burkholderia pseudomallei. The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for B. mallei; moreover, it is a suitable method for differentiating between Burkholderia mallei and Burkholderia pseudomallei. This technique can detect Burkholderia mallei in a short time with high precision and sensitivity.

Clostridium novyi (C. novyi) causes deadly Black disease in sheep and rarely in other animals. Alpha toxin (α-toxin), the most apparent pathogen of this disease, is produced by C. novyi type B. Economic damages of C. novyi include sheep mortality costs, depreciation of affected farms, and health problems with infected carcasses. The identification of C. novyi and isolation of its pathogens by conventional methods is a time-consuming process, necessitating a simple and rapid method for isolating and detecting pathogenic C. novyi. Therefore, this study aimed to molecularly identify α-toxin in local C. novyi isolates from the sheep livers. In this study, 75 livers suspected of Black disease were sampled. The samples of the liver were cultured under anaerobic conditions. Some of the cultured colonies were used in biochemical tests. For molecular confirmation, the DNA of isolates was extracted, and the isolates were confirmed by the polymerase chain reaction (PCR) on the liver tissue and cultured samples using specific α-toxin primers. The PCR on α-toxin produced a band in the range of 609 bp, indicating that the samples belonged to C. novyi. According to the results, of 75 isolates, 18 isolates were confirmed as C. novyi. C. novyi type B was isolated from the liver and confirmed by biochemical and molecular characterization. The PCR assay ensured a sensitive and specific tool for the detection of C. novyi in the samples.

Mycoplasma agalactiae (M. agalactiae) is known as the main etiological agent of contagious agalactia (CA). The CA is a disease affecting dairy sheep and goats, the main characteristics of which include keratoconjunctivitis, arthritis, and mastitis. This pathogen results in milk production reduction and suppression, thereby leading to serious economic loss. In the present study, 125 sheep and goat samples were collected from 15 provinces of Iran. Cultural and molecular methods were used for sample characterization. After extracting genomic DNAs using the phenol/chloroform method, the PCR technique was employed to detect Mycoplasma genus in 163bp fragment of 16S rRNA gene (M-PCR) and M. agalactiae in 800bp fragment of conserve and specific P30 lipoprotein gene (P30-PCR) in cultural and clinical samples. Finally, to validate the experimental approach, a 375 bp amplicon of P80 lipoprotein was amplified using the MA-PCR. Out of 125 samples under investigation, 43 cases were positive, and Mycoplasma colonies were observed in the pleuropneumonia-like organisms agar culture. Based on the results of the M-PCR method, 61 specimens (out of 125 samples) were scored positive for Mycoplasma presence. Furthermore, 20 samples were positive according to the P30-PCR data. It should be mentioned that the MA-PCR was performed based on the P80 gene on 125 total samples to furtherverify the results for M.agalactiae detection. Based on the obtained data, P30 and P80 genes were presented and amplified in all Iranian M. agalactiae isolates (n=20). Our results indicated that the P30 gene was conserved and specific to all Iranian M. agalactiae isolates and this new P30-PCR method (as an MA-PCR technique) might be useful in the detection of this pathogen.

The purpose of this study was to evaluate the influence of clinical, physiological factors (age, sex, history ofabortion) and environmental factors (marsh or swampy environment, the climate, the distribution of mosquitoesin the environment, altitude above sea level) on the Blue tongue virus seroprevalence in the sheep flocks. Thisstudy was performed on 200 blood samples were randomly collected from 19 herds and 7 villages in the KHOYcity. Sera was investigated using by ELISA to determine serum levels of antibodies against the Blue tongue virusand the results were analyzed statistically. Information required in questionnaire forms were collected during thisstudy. Results showed from a total of 200 samples 74 cases had clinical symptoms such as rhinitis, stomatitisand laminitis which only 26 samples were seropositive. A total of 40 male 23 cases (57/50%) and from the 160females 111 cases (69/37%) had a positive ELISA test that there was no signifcant difference between male andfemale animals(P>0.05). There were 66 samples with abortion history of the ewes trap frequency of positiveserum samples that 49 samples were found positive with abortion which is equivalent to 14/44% was achieved.Different age groups had signifcant difference more susceptible to infection with BTV antibody (P<0.05).Morethan 80% of positive samples belonged to swamp and semi-swamp environments and other of samples related tonon-swampy areas. Among the positive serum samples more than half of the samples were with hot and humidweather. The based of distribution rate of mosquitoes in areas with many mosquitoes more than 40% of positivecases were scattered about in too. The altitude above sea level in low-lying areas close to half belongs of thepositive samples and post lowland (altitude 1,000 m). Results of this study is shown some factors such as age,history of abortion, swampy environments, heat and moisture, many distribution of mosquitoes and low-land areascan be risk factors of sero-prevalence of BTV among sheep and should be ways for control of sero-prevalence ofBTV and prevention from change to clinical form of disease.

The aim of this study was determining the prevalence rate, serotyping and sensitivity to antibiotics of Salmonella isolated from slaughtered broiler chickens of Guilan province. Two hundred carcasses were randomly selected out of 20 slaughtered broiler flocks and then 1000 samples were collected from different parts of carcasses (skin, meat, gallbladder, liver and intestine). All samples were cultured in different media, and the isolated Salmonella serotypes were identified. Sensitivity and resistance of isolated Salmonella were determined to 15 antibiotics. Three flocks (15%) and in 8 cases (0.8%) were positive to the Salmonella. Two serotypes were identified, including 6 samples (75%) of Salmonella enteritidis and 2 samples (25%) of Salmonella thompson. Salmonella was isolated from 4 samples of liver (50%), 2 samples of skin (25%) and 2 samples of caecum (25%). In this study, no salmonella was isolated from poultry meat. The results of sensitivity and resistance of Salmonella to antibiotics indicated that, 100% of isolates were resistant to cefazolin, streptomycin, kanamycin, tetracycline, nalidixic acid and sulfonamide + trimetoprim. There were three resistance patterns among the isolates, 100% of isolates were resistant at least to seven antibiotics and 25% were resistant to eight antibiotics from fifteen antibiotics. It is concluded that, the prevalence rate of Salmonella infection was lower than to the other studies, the dominant serotype was Salmonella entritidis and 100% of isolates were sensitive to ceftriaxone, gentamicin and chloramphenicol.

NDV (Newcastle Disease Virus) is one of the viruses that cause disease in avian with severe economic losses in the poultry industry in many countries. Fusion protein (F) which plays a major role in the virus pathogenicity contains several regions that have a role in the fusion process. Mutation in the sequence of HR1 & HR2 regions of this protein prevents fusion of the virus to host cell. In addition, the proteins of HR1 and HR2 regions have antitumor properties that are related to their pathogenicity. In this investigation we used Newcastle disease virus NR43 isolate, from poultry diseases diagnostic department of Razi vaccine and serum research institute. RNA was extracted using SDS and proteinase K procedure. In the next step, RT-PCR was carried out and then cDNA cloned in pTZ57RT vector. After sequencing and alignmenting of the cDNA, a pair of proper primers for cloning HR1 in expression vector Pet32a(+) was designed. The HR1 expression was carried out by SDS–PAGE Western- Blotting in which the peptides were blotted onto nitrocellulose membrane using Ni-NTA anti His tag (1:1500 dilutions) coupled to HRP enzyme. A peptide with 23.76 kD/a molecular weight s peptide was obtained. By cloning and expression of HR1 region of protein F, it will be possible to express the whole gene that could be introduced as a novel vaccine against NDV.

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.

Experimental oil-emulsion vaccinc \Vas l'onnulated \Vith a ratio of one part of 0.05% ~-propiolactonc inactivatcd antigcn and l'our parts oil adjuvant ISA-70. Four hundn.:d whitc Ily-Linc (\\'-36) chicks were rearcd in t'our groups. Groups 1 and 2 \Vere vaccinated b) live attenuated 1·1120 at day 10 and reared separately from groups 3 and 4. At weck 16 groups 1 and 2 were received experirnental oil-emulsion and commercial oil-emulsion vaccines respectively. At this time. group 3 \Vas n:ceivcd single dose of experimental oil-emulsion vaccine and group 4 \\"s unvaccinated conlrol. At \Vcck 29 ail or groups \\cre challenged by 0.25' I07r': ID,,/bird or intCcted allantoic Iluid whcn the)' were on peak of production. Ali groups were bled fi-cl]uently and the sera were assayed by ELISA and ACJI J) tests. Clinical signs and high percentage drop in egg production in groups 3 and 4 v.ere notcd. Moreover. no virus and/or viral antigen in the trachea or groups 1 and 2 \Vere dctcctcd at days 5 and 10 postchallengc. The results or clinical observation cgg producl ion. virus isolation and delcction in Ihe trachea and levels of antibody suggcsted Ihal layers vaecinated wilh a combinalion of live allcnuated and the cxpcrimcntal inactivalcd vaccines had Ihc highcsi proteclion.

By emergence of very virulent infectiolls bursal disease virllses (vvIBOVs), ail classical measures were called into question. In this study an inactivated IBO vaccine with bursal and embryo origins using a recent local vvlBOV isolate, 1R499, was prepared. The virus cultivated in both SPF emberyonated eggs and chicks to evaluate the efTects of host system on immunogenicity of the vaccine. The results showed that inoculation of broiler breeder chickens with the inactivated IBO vaccine at 20 weeks of age produced significantly greater levels of antibody in comparison with IBO live vaccine and induced antibody titers with a lower variation than the other. The mean antibody titer raised by bursal origin vaccine was significantly greater than other inactivated vaccine and it persisted throughout this study. Our result show that inactivated vaccine with bursal origin induces high level of antibodies, which is essential for protecting of the birds against the vvIBOV strain. Also il shows that incillding of very virulent strain in inactivated vaccine provides bcttcr protection against lield vvlBOV strains.

Tht: clinical signs and postmortem lindings including rt:spiratory system distress and Icsions in upper respiratory tract wert: observed in 17 commercial layer flocks l'rom differcnt parts of Iran. Laryngotracheal tissues l'rom the alTeetcd birds with suspectcd inlectious laryngotrachitis (lLT) wcrc examined l'or deteetion of the causative agt:nt using virus isolation (VI), agar gd imlllunodilTusion (AGIO), histopathology (HP), and polylllcrase chain reaction (peR). 12 (70.5%) of the salllples were formed typical poeks on chrioallantoic membrane of embryonated specilic pathogen free eggs in VI method. By AGIO test Il (64.7%) of the samples \Vere detectcd as II.T virus (1I.Tv) that gave c1ear lines \Vith hyperimmune IL T serum. Of the samples 7 (41.1 %) were illustrated lesions chara\.:teristics of ILT including inliltration of inllammatory œlls and syncytia formation. In 14 (82.3%) or the salllples ILTv was dcteetcd h) l'CR. The high positive perccntages of the l'CR indieate that the technique is applicable in rapid diagnosis of ILT for its accurucy, sensitivity and specilieity.

To determine the type, severity and frequency of gross, histopathologic changes and tissue tropism of avian influenza virus (AIV) a group of twenty, 5-week-old chickens (hatched from SPF eggs) were inoculated intravenously (IV) with type A AIV [AiChicken/lran/259/1998(H9N2)]. Another twenty chickens were inoculated IV with sterile chorioallantoic fluid (CAF). Tubulointerstitial nephritis and pancreatitis were the most frequent specifie histopathologic changes. Influenza nucleoprotein was demonstrated in renal tubule epithelium and in acini ofpancreas/foci ofnecrosis in both organs .The virus was localized in cytoplasm and nuclei of proximal/distal tubule epithelium. Common nonspecific histopathologic changes were Iymphoid and reticuloendothelial cell hyperplasia in spleen, leukocyte cell infiltration in myocardium and lymphocyte infiltration in Iiver. The results indicate that the low pathogenic AIV isolate was epithliotropic in chicken.

Based on primers from gl glycoprotein, a polymerase chain reaction (PCR) assay was optimized for detection of bovine herpes virus type 1 (BHV -1). A 468bp DNA fragment ofnine isolates was amplified. The PCR products were detected by ethidium bromide staining after gel electrophoresis and confirmed by sequencing. The nucIeotide sequence alignments revealed a highly conserved region in gI gene within the isolates. Results suggest that PCR can be used as a reliable diagnostic tool for rapid detection of BHV -1 infections.

Ornithohac/erium rhino/rachea/e (ORT) is a relatively rceently diseovered bacterium. which it is associated with respiratory diseases, Tracheal samplcs from carcasses of 100 roultry tlocks submitted to Razi Institute were examined for ORT isolation. Ali these tlocks were affected with respiratory disorders. Colonies of ORT were detected after 24h of incubation and identification of ORT isolates were carried out based on the biochemical and serologie al charaeteristics. Fitiy-nine isolates From broilers, broiler breeders, and layer Ilocks were idcntilied as ORT and analyzed for serotyping. The isolates were identified as serotype A. using standard • antisera againsl ORT anligens in rapid slide agglutination, agar gel precipitation and immunodot tests. The identification of the species was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis of whole cell proteins. High similarity ofprotein profiles of the isolates was observed. These preliminary results establish that ORT serotype A is associated with outbreaks ofrespiratory diseases in commercial poultry.

Î II prell.:nt against reecnt virulent strains or in/ectious bursal disease virus (IBDY), a local isolaLe of the virus was propagated in bursal tissue, l(lfIllali/.ed and used I(lr vaccine production. An experimental vaccine adjll\anted hy oil ISA-70 was prepared and compared with a commercial IBDY-NDY inactivatcd vaccine. A single injection or the two vaccines proteeted chickens against mortality hut the oil adjuvanted bursal derived vaccine conli:rred a higher percent or bursal protection.

An out break of infectious laryngotracheitis (lLT) in a large tlock following vaccination against IL T is described. Laryngotrachcal samples were obtained from sorne of infected pullets. Isolation and characterization of agent virus were carried out on chorioallantoic membrane (CAM) of embryonated specifie pathogen free (SPF). Several pocks on CAM were observed after live days of incubation. The isolated virus was neutralized by mono-specifie antiserum against vaccinal IL T virus. For evaluation of pathogenicity of the isolated virus twenty 8-week-old SPF chicks were eye drop inoculated with 103 and 104 EID;o% virus respectively and observed upto 7 days. Only two chickens in group 2 were shown moderate signs including conjunctivitis and swelling of lower eyelid. Histopathologic features in tracheal of both infected pullets and experimental SPF chicks were epithelial hyperplasia, multinuclear giant cells (syncytia) with eosinophilic intranuclear inclusion bodies and infiltration of intlamatory cells, which was milder in latter. These findings indicate that the isolated virus was similar to vaccine strain. Our study suggests that under improper IL T vaccine administration and generally bad management practice conditions, the IL T may occur following vaccination.