Archives of Razi Institute
Volume:55 Issue: 1, Winter 2003

The molecular detection of avian infectious bronchitis virus by use ofRTPCR and multiplex nested PCR in Fars province of Iran was investigated. Detection has been done on tracheal swab s:!mnles of 30 broiler tlocks in age of 7-8 weeks. Flocks were selected from nIU~' broilers rising regions orthe province. Detection was performed by primers specifie for Massachusett, 4/91 and 0274 serotypes. In this study 16 samples were positive for IBV 4/91 typeand 1 for Massachuset type. One swab sample showed a mixed infection of those serotypes. 0274 serotype has not been detected in this study. This is the first report of the presence and a prevalence of IBV type 4/91 in Fars province.

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.

The recombinant glycoprotein 0 (gO) of herpes simplex virus type-I (HSV -1) in baculovirus expression system was produced. Spodoptera Frugiperda cell, clone 9 (Sf9) was cultured in modified Grace's medium and inoculated with 5- 7 multiplicity of infection of HSV-I recombinant baculovirus carrying gO gene. Inoculated cells were harvested 96h post inoculation and treated with 3- [(3-cholamidopropyl)dimethylammonio ]-I-propanesulfonate (CHAPS) and phenylmethylsulfonyl tluoride (PMSF) and sonicated at 20KHZ for 5 times. A western blotting was developed and applied to detect the prepared protein. Three groups of BALB/c mice each of 7 mice were inoculated with the recombinant gO, sublethal dose of challenge virus (104STCIDso) and PBS respectively. Ali inoculated mice were challenged with IOMLOso (106sTCIDso) of the wild IISV-1. Ali mice who had received the recombinant gO survived while 14.3% and 71.5% of mice inoculated respectively with either sublethal dose orthe virus or PBS died.

VI' 1 protein of foot-and-mouth disease ViruS (FMDV) contains the immonogenic hypervariable region of the virus. The antigenic variation in FMDV is particularly related to the difference between nucleotidc and amino acid sequences of capsid protein. On the basis of this phenomenon. type diagnosis of FMDV can be done by the polymerase chain reaction (PCR). In order to specifically identify the 0, FMDV serotype of Iran the complete coding sequence of its VPI prote in was amplified by RT-PCR, and nuclcotidc and amino acid sequences of the PCR product were dctermincd. The nuclcotide and deduced amino acid sequence exhibited 84% and 88% homology with the VP 1 region of serotype O,K. respectively.

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.

ln order to develop an ELISA kit for detection ofanti-DNA antibodies several procedures were examined. In this study the best method for native DNA preparation, solid phase preparation, formulation of buffers, design oftest performance, and a storage condition of the kit components was introduced. DNA extraction was performed by phenol procedure and purified one more time to lose protein impurities. To prepare the coated micro plate, a precoating stage using poly-L lysine to enhance DNA attachment and an extra post-coating stage for neutralizing its negative charge were applied. To detennine the sensitivity and specificity, 120 serum samples were simultaneously tested with the developed and commercial kits. The results indicate that sensitivity and specificity of the developed kit is 97% and 100% respectively with 99% accuracy and 5% coefficient of variations. Moreover periodic examinations on kit components reveal that the kit is stable at least one year in 4°C without diminished quality.

The prevalence of subclinical 10hne's disease was investigated in 293 (97 male and 196 female) cattle slaughtered in Urmia abattoir during year 2001. Samples were included both faces and intestinal epithelial tissues which collected from the area between ileum and cecum. Zeihl Neelson staining method was applied to diagnosis the acid-fast microorganism (AFM) as an indication ofsubclinical 10hne's disease. AFM was found in 9 of293 samples (3.07%). Females with AFM (3.57%) were aged over 2 and males (2.06%) over 3 years old. There were no difTerences between sex and age variation. Distribution of subclinical 10hne's disease in spring, summer, autumn and winter were 3, l,land 4 cases, respectively indicates that the difTerence (P<0.05) in seasons was statistically significant. The results indicate that subclinical 10hne's disease could be a problem in cattle either male or female.

The Interferon gamma (IFN-y) gene 1 was isolated from phytohemagglutinin stimulated peripheral blood mononuclear cells of a healthy individual blood donor using RT-PCR technique. The gene was cloned under the control of the different promoters' expression vectors such as pET32a (Novagen), pQE30 (QIAGEN), pSKA-IBA (Strep-Tag), pRSET (Invitrogen), and then expressed in E.coli. The transcription of IFN-y mRNA was determined by northem blot analysis. The level of expression of human IFN-y in pRSET vector under the control of T7 promoter was determined by laser-based densitometry of SOSPAGE and found more th an 26% of total bacterial protein. The expression was confirmed by western blotting. The expression of IFN-y under the control of promoters of pET32a, pQE30 and pSKA-IBA plasmids was detected by SOS-GAGE and western blotting.

The use of accu rate, rapid, cheap and sensitive methods for measurement of cytokines in body fluids is absolute prerequisite to detine involvement of these mediators in various clinieal situations and pharmacological effect of recombinant cytokines administration. In this study a sandwich enzyme-linked immunosorbent assay (ELISA) was designed for detection of small amounts of human interferon gamma (hIFN-y). Alter immunization of a female New Zealand White (NZW) rabbit and a female BALB/c mouse against recombinant human IFN-y, a high level titer of antibody was produced that confirmed by dot blot technique. A precipitated antibody from rabbit serum was used as tirst antibody and mouse serum used as second antibody. By determination of the best antibody concentrations and optimization of other conditions, an ELISA system was designed. The data indicate that this ELISA was efficient and sensitive for detection of as little as 40nglml of recombinant hIFN-y.

For rapid c1inical diagnosis and also determining the immune response of healthy persons to whooping cough a stained pertussis antigen was prepared. In this regard the 48h culture of Bordetella pertussis, Tohama wild strain, on methyl cellulose enrichment medium (B2) was used. The medium was supplemented with methyl cellulose, Rosebangal as vital color and phosphate butTer containing Thiomersal (1/5000). A comparative study with Bordetella pertussis/toxin IgG ELISA has shown that the local antigen is highly specifie and sensitive. Therefore the antigen may provide a diagnostic laboratory tool in epidemiological study.

During six months, Oct to Mar 2000, 300 cases of condmned bovine kidneys were randomly submitted to the pathology laboratory for diagnosis ofrenal diseases. The macroscopical and microscopical features of various types of bovine renal diseases were discribed. The frequency rate of macroscopical lesions was included 6 renal cysts, 7 hydronephrosis, 3 pylonephritis, 165 white spotted kidney, 82 large pale kidney, 15 small shrunken kidney,4 congestion, 3 pale infarction and 15 without any lesions. Histopathologic study of these cases with H&E stain, revealed that the important lesions were occured in interstitial tissue of kidney. The frecquency rate of microscopie lesions was included 6 renal cysts, 7 hydronephrosis, 3 pyelonephritis, 165 acute multifocal or diffuse interstitial nephritis, 80 nephrosis, 1 glomerulonephritis, 1 amyloidosis, 15 nephrosclerosis, 4 congestion, 3 infarction and 15 without any lesions.

From Mar 2000 to Nov 2001 the prevalence of resistance to ampicillin, sulfomethaxazol-trimetoprim, (SXT) nitrofurantoin, nalidixic acid, ciprofloxacin, cephalothin, and gentamicin in 740 gram-negative bacilli isolated from outpatients with acute urinary tract infections (UTIs) at Kashan Central Laboratory was prospectively evaluated. Eschericia coli (Eco/i) (75%) was the most common causing UTIs, followed by Klebsiella spp. (17%) and Proteus spp. (2.1 %). Arnong them 80% more isolates were resistance to ampicillin and 47% more isolates to SXT. Cephalothin resistance among Ecoli isolates was >28%, Klebsiella spp 32.1 % and Proteus spp. 40%. Overall, the rate of ciprofloxacin resistance among them was 7.8 to 18.2%. Nalidixic acid resistance among Ecoli isolates was 6.5%, Klebsiella spp 9.7% and Proteus spp 15%. Arnong the isolates, 15% less isolates were resistance to gentamicin. Nitrofurantoin showed the lowest resistance rates «4.5%) with exception of the Proteus spp. (10%). The high prevalence ofresistance is surprising, it may reflect the widespread use of antibiotics in Kashan continued regional, and national surveillance is warranted.

Polioencephalomalacia is none infectious neuro degenerative disease that affect sheep, goat, deer and cow. Polioencephalomalacia commonly occur in cattle fed ration rich in carbohydrates with \ittle roughage that may changes microflora of rumen and impaired synthesize of thiamine (Thomson et 0/200 1, Boyd & Walton 1977). Polioencephalomalacia could represent a multifactoral metabolic disorder with multiple etiologies such as nutritional problem, high level of carbohydrate, water deprivation, salt intoxication, high level intake of sulfur, renal encephalopathy, amprolium and sorne plant such as Bracken fem and Horsetail. Probable mechanisms that may cause thiamine deficiency polioencephalomalacia in ruminant are disorder of absorption, synthesize and destruction of thiamine by thiaminase (Radostit et al 1994, Bakker et 0/1980, Jeffrey et 0/1994, Loew & Dunlop 1972, Olkowski 1992, Thomber et al 1979). Evidence (or theories) linking thiamine with the ruminant polioencephalomalacia disorder incIude cIinical response to thiamine injection in sorne individual (Thomson et 012001).

8irds are routinely exposed to this fungus and only rarely become pathogenic and the lower respiratory tract is most severely affected by inhalation rout. There have been sorne recent reports about respiratory aspergillosis in ostrich (Kyoung 200 l, Marks et al 1994). Aspergillus spp usually cause disease under condition of stress, immunosupression, and prolonged treatment with antibiotics or massive exposure to the microorganism. Clinical signs of aspergillosis in ostriches are weight loss, lethargy and dyspnea (Kyoung 2001). Other less common forms of aspergillosis in birds are encephalitis, ophthalmitis, osteomyelitis, dermatitis and systemic form (Fitzgerald & Moisan (1995). AJumigatus was isolated in sorne cases of respiratory aspergillosis (Campbell (1986). It has been reported that aspergillosis was observed in ostriches in the late 19th and early 20th centuries, but is relatively uncommon today (Terzich & Vanhooser (1993). This is the tirst case report of aspergillosis due to AJumigatus in Iran. The ostriches ofthis report had received chloramphenicol and enrofloxacin for a long time (45 days) so these drugs may have been the cause of the pulmonary aspergillosis.