[Detection of specific antigens of Newcastle disease virus using an absorbed Western blotting method]

Newcastle disease virus [NDV] is an economically important poultry pathogen with a worldwide distribution that may infect a wide range of domestic and wild avian species. The identification of different pathotypes of NDVs plays an important role in the diagnosis and development of vaccines to control and eradicate NDV infections. In our previous study, we showed that mono-specific antibodies can differentiate velogenic and lentogenic strains of NDV in Agar Gel Immuno-Diffusion tests. To evaluate the ability of the specific antibodies to detect NDV specific antigens, this study was conducted with a range of NDV isolates. The samples included 9 NDV neuropathogenic/velogenic isolates from diseased chickens collected from poultry farms in central and northern parts of Iran plus La-Sota and B1 vaccine strains. All samples were propagated in embryonated chicken eggs and concentrated and purified by ultra-centrifugation. All samples were subjected to 12.5% SDS-PAGE and Western blotting using the specific antibodies mentioned previously. In SDS-PAGE all velogenic and vaccine strains showed the same electrophoretic pattern. The detected bands included 15, 38, 46, 48, 53, 55, 68, 74 and 220 kDa proteins. In Western blotting analysis, the mono-specific antibodies reacted specifically to the viral proteins with 15, 38, 48, 55, 74 and 220 kDa and non-specifically to the viral protein with 53 kDa. The results suggest that specific anti-NDV antibodies can react specifically to glycoproteins [haemagglutin-neuraminidase and fusion proteins] but not to internal proteins [nucleoprotein or matrix protein] of NDV strains

Genetic and phylogenetic analysis of the ribonucleoprotein complex genes of H9N2 avian influenza viruses isolated from commercial poultry in Iran

The H9N2 subtype of avian influenza viruses [AIVs] has been isolated in multiple avian species in many European, Asian, African and American countries. Since the first outbreak of H9N2 virus in Iran in 1998, this virus has widely circulated throughout the country, resulting in major economic losses in chicken flocks. Several amino acids in the virus ribonucleoprotein [RNP] complex including the nucleoprotein [NP] and polymerase [PB2, PB1 and PA] proteins are associated with host range and virulence. Our aim was to understand the molecular characterization of RNP complex proteins of Iranian H9N2 subtype isolates. The full length nucleotide sequences of RNP complex genes of two strains designated as Ck/IR/ZMT-101/98 and Ck/IR/EBGV- 88/10 were amplified and sequenced. The phylogenetic analysis revealed that both strains were located in different sub-lineages. However, based on the genetic similarities, PB1, PA and NP genes of Ck/IR/EBGV-88/10 strain had a close relationship with a H7N3 subtype strain, isolated from Pakistan. Most positions of RNP proteins contained amino acids typical of avian determinants of host range. The results showed that the Iranian RNP complex genes have undergone genetic reassortment. Continuous AIV monitoring in poultry industry would help to obtain more information about genetic variation of H9N2 viruses and possible emergence of virulent and/or pandemic viruses

[Molecular epidemiology of foot-and-mouth disease virus in ruminant during 2005-2006 in Iran]

Foot and mouth disease [FMD] is a highly contagious viral disease of ruminant which causes fever and postule on mouth, hoof and teat. The main purpose of this study was to determine and charachtrize isolated FMD virus from Iran between 2005-2006, and to compare it with vaccine virus strains. After preparation of samples, serological test for typing of virus was performed. In order to virus isolation. the samples were inoculated to IBRS2 cell, RT-PCR and PCR were used for sequencing. Two dimentional virus neutralization test was carried out for detecting of immunological relationship [r value] between the field isolate and virus presented in vaccine. Detected strains were as follows: 241 samples of type AO5IR, 125 of type A87IR, 79 of type 0.3 of type Asia and 714 negative out of 1162 samples. Average r-values of type A, 0, Asia field virus with vaccine strains were 50-92% and 97%, respectively. Phylogenic tree was designed according to the nucleic acid sequencing data. There is not strong relationship between field viruses of type A and vaccine viruses. However a strong relationship was shown for type 0 and Asia ones with vaccine virus strains

BVDV induced gastro-neuropathy outbreak in a feedlot calves around Tehran [Iran]

An outbreak of a lethal disease was reported in 4-6-month-old Holstein calves in a feedlot around Tehran. The signs of central nervous system and gastrointestinal system [GI] involvement were observed in the diseased animals. Necropsy samples of GI, liver, kidney, spleen and lung from 3 died animals were prepared for histopathological examination. Blood and formalin-fixed ear notch samples of 6 calves were submitted for RT-PCR, antigen-capture ELISA [ACE] and immunohistochemistry [IHC] for the detection of BVDV. The results of ACE on buffy coats were negative but RT-PCR of all 6 cases and IHC of 4 cases were positive for BVDV infection. Based on the clinical signs and pathological findings in the GI system and brain, we strongly suggest that the BVDV may represent a gastro-neuropathogen strain. To the best of our knowledge, this is the first outbreak of gastro-neuropathogenic BVDV infection in Iran, which may be acquired postnatally

prevalence of Escherichia coli O157:H7, Listeria monocytogenes and Campylobacter spp. on bovine carcasses in Isfahan, Iran

This study was carried out to determine the prevalence of foodborne pathogens, Escherichia coli, E. coli O157:H7, Listeria monocytogenes and Campylobacter spp. on slaughtered cattle in Isfahan, Iran. A total of 203 cattle carcasses were sampled by surface section of neck meat taken immediately after slaughter and analyzed using microbiological examinations. Suspected colonies to E. coli O157:H7 were confirmed by a specific polymerase chain reaction method [PCR]. The results showed that the contamination rate of samples to E. coli and E. coil O157:H7 were 42.4 and 6.4%, respectively. Seasonal distribution showed that the highest prevalence of E. coli and E. coli O157:H7 occurred in summer samples. Six carcasses carried L. monocytogenes whereas Campylobacter spp. were not detected on any carcasses. The results indicated that prevalence of E. coil and E. coli O157:H7 was high on bovine carcasses in lsfahan. This condition should be considered as a probable hazard for human health

Comparison of ear notch immunofluorescent, buffy coat antigen-capture ELISA and RT-PCR for detection of calves suspected to actuely infected with bovine pesti virus

Twelve calves of 4-10 months old with clinical signs suspected to bovine viral diarrhea virus [BVDV] infection were selected for this study. Histopathologic sections were performed on formalin fixed paraffin-embded ear notch biopsies, mounted on poly-L-lysine coated slides and stained for BVDV by Anti-BVDV monoclonal antibody labelled with fluorescein isothiocyanate. Stained sections were examined by fluorescent microscopy for detection of green fluorescent evidence within the cytoplasm of keratinocytes, other dermal cells and chondrocytes. Detection of BVDVantigen in buffy coat cells was performed by using a commercially available antigen-capture ELISAkit, and RT- PCR, using a universal primers set, specific for all pestiviruses. Based on 11 positive cases detected by RT-PCR, immunofluorescent staining and antigen-capture ELISAcould detect 8 cases [72.72%] and one case [9%] respectively. Results of this study suggest immunofluorescent test on ear notch biopsies has a relatively high sensitivity, and can be used as a reliable and feasible method for detection of calves with acute infection with BVDV

[ survery on frequency of foot-and-mouth disease virus carriers of slaughtered cattle in Zyaran abattoir]

To determine frequency of FMDV carriers in slaughtered cattle at Zyaran abattoir. Cross-sectional study. Three hundred and one tonsils of clinically normal cattle. All of tonsils were tested for determination of FMDV genome using RT-PCR. Then, 30 samples were cultured on MDBK. 20 samples had positive findings in RT-PCR test. Based on sex, age and breed of cattle, RT-PCR results compared with Chi-square and Fisher's Exact Test. McNemar test was used for comparing of virus culture results with RT-PCR. Ninety nine samples were positive in RT-PCR. Relative frequency of FMDV genome presence in tonsils of clinically normal cattle had a significant difference based on sex and breed. The Sistani and Holstein bred had the highest and the lowest relative frequency, respectively samples on cell serotype O of culture were FmDV. One positive sample [Asia 1] Two positive sample[Asia1] was shown by ELISA. Total relative frequency of positive FMDV genome [32.9%] indicates the extensive exposure to FMDV. Breed variation among Sistani cattle [as a variety of Bos indicus] and other bred should be studied in a controlled condition

Identification of the foot and mouth disease foci from susceptible foci in Khorasan Razavi province

This study was conducted in Khorasan-Razavi during 23 months [since February 2004 to December 2005]. The samples [83 samples] of suspected farms were collected from tongue and mouth epithelium and tested by indirect sandwich ELISA and RT-PCR. During the study, twelve foci during the year 2004 and 23 foci during the year 2005[up to December] were approved. Maximum distribution of FMD foci was observed in spring [63%]. Pearson's correlation coefficient and Chi- square tests were carried out for data analysis. Attention was paid to frequent traffic of farm in springs since there is an increase of the disease in this season. There was no correlation between the sheep density and the disease must be account in cattle. However, with respect to considerable sheep population in Iran and its role in persistence of the virus, the vaccination of sheep seems to be essential for the control of FMD

Study of effector antigens of IBR virus on humoralimmunity

The objective of this study was to determine the protein pattern and antigenic structure of BHV-1. Ten viral isolates from cows with clinical signs of respiratory infections were selected for this study and confirmed by SN test. All samples were cultured on cell cultures and purified by ultracentrifugation. Purified isolates were electerophorsed using SDS-PAGE method and immunoblotted. Viral antigens were detected using anti BHV-1 bovine antiserum and molecular weight of the bands were determined. All samples showed identical band number and molecular weight except for one sample which revealed an additional 120-125 KDa band. Also immunoblotting data resulted in identified of twelve bands [150, 130, 115, 97, 77, 70, 55, 45, 40, 38, 32 and 25 KDa] suggesting involvement of these antigens in evoking humoral immune response in affected cattle

Detection of bovine Leukemia virus antigens expressed in lymph node tumors that induce humoral immunity in cow

Detection of bovine leukemia virus antigens can induces humoral immunity in cow were evaluated. Fifteen lymph node from infected cows that had positive results in AGID and ELISAtests for BLVand have clinical signs of lymphosarcoma, in addition five lymph node from apparently healthy cows that had negative results in serological tests. After preparation of extract of lymph nodes, all of the samples electerophoresed in SDS-PAGE system in discontinuous 5 and 10% gel. All gels transferred to nitrocellulose membrane in Biorad blotting system. Antigens were detected by BLVpositive antisera by using HRPO conjugated protein G and Tetramethyl benzidine as substrate. At least 25 proteinal band were detected in SDS-PAGE of tumoral and normal lymph node. In tumural tissues two additional band 24 and 51 kda were detected. In western blotting of those samples, gp51 antigen were detected in all tumoral lymph nodes, p24 antigen were detected in 5 samples from 15 samples and in non of apparently healthy samples non of those two antigens did not detect in WB test. These results were shown that gp51 were expressed in high level in tumors and induced a strong humoral immune response but p24 is a weak and non-common antigen in lymphatic tumors. Gp51 is most important and first antigen in all of the cases that infected by BLV

Detecting the P24 antigen in lymph nodes of BLV infected cattle

In this study 19 BLV infected and 2 healthy lymph nodes were analysed by Western blotting to detect viral p24 antigen. Western blotting was carried out by Rabbit anti p24 serum and p24 monoclonal antibody. Rabbit serum in western blotting could detect 45, 50, 54, 58 and less than 19 kD bands of proteins and monoclonal antibody could detect 30, 32, 39, 45, 50 and 53 kD bands. Our results indicate polymorphism of proteins with p24 epitope in different BLVinfected samples. Otherwise, it could be concluded that BLVstructural proteins sharing p24 epitopes have high degree of phenotypic diversity

Molecular diagnosis of Marek's disease virus [MDV] in Iran

The purpose of this study was diagnosis of Marek's disease virus as one of the causative agents for visceral tumors in chickens using Polymerase Chain Reaction. Forty blood samples from the chickens without any clinical signs of Marek's disease and another 42 tumoral tissues from commercial chickens were collected. The whole DNA of the samples were extracted using a silica gel DNA extraction kit, then PCR test was performed using specific primers detecting 132bp tandem repeat and antigen A gene of MDV, finally electrophoresis of PCR products was done in Iko[3]/41 agarose gel. No positive results were obtained in blood samples for MDV and it's vaccinal strain, but about 47.6% of samples were positive. The tumoral tissues including liver, spleen, proventriculus, ovary, breast muscle and bursa of Fabricius. The vaccinal strain of MDV [Rispens] was not detected in any of examined blood samples, as the period of viremia for this virus is very short. Serotype 1 of Marek's disease virus was detected as a causative agent of tumors in the chicken farms of Iran as a first step in this study

Study of Infection caused by pseudorabies virus [PHV-1] in Iranian wild boars [First report]

Serological study of infection caused bypseudorabies virus [PHV-1] in Iranian wild boars. Descriptive study. 28 wild boars were captured in differentprovinces of Iran. After bleeding of hunted boars, serum sampleswere collected and tested by using a blocking ELIS A kit [Eurodiagnostica]. Results were detected by an ELIS Areader machin. 12 out of 28 sera samples [42.8%] from the boarsof Tehran. Markazi, Khorasan, Zanjan, Golestan, Ears andEsfahan provinces were positive. Total infection rate [42.8%] was diagnosed as 40% of male versus 50% offemale boars. Average age in positive and negative caseswas 5.1 and 4.9 years, respectively. This results show theconsiderable spread of the infection among Iranian wildboars [42.8%] which is indicating a need of great attentionsto the risk of transmission of the disease to animals, humanand specially boar meat consumers

[Designing of a specific immunofluorescence method for differentiation of velogenic and vaccinal strains of newcastle disease virus]

To Prepare of a specific FITC conjugate antibody for differentiation of velogenic and vaccinal strains of Newcastle disease virus. Experimental study.27 rabbits.4 velogenic strains of Newcastle disease virus were obtained from collection of viruses in Virology Laboratory of Faculty of Veterinary Medicine and two vaccinal strains [Bl and Lasota] were propagated in embryonated eggs and purified by ultra centrifugation. Purified viruses used for immunization of 7 groups of rabbits, each consisting of 4 animals. Each group was immunized by one of the virulent or vaccinal strains and one group by mix of vaccinal strains. The immunization process took about 4 months. Sera samples fro immunized animals after absorption by each of the vaccine and velogenic strains were put in proximity to one another in Agar gel Immunodiffusion test. Specific antibodies conjugated with FITC. 28 velogenic isolates, 2 vaccinal strains and 14 negative samples were tested by using the conjugated specific antibody.Eventually only one precipitate line was observed. That was indicative of the fact that specific antibody against velogenic and vaccine strains was obtained. The produced specific antibody can detect unique viral antigen and respond against it. This specific FITC conjugated antibody can differentiate velogenic and vaccinal strains of NDV in shorter time than classic methods

[Study on electrophoretic proteinal pattern of lymph nodes of cows with bovine leucosis and comparison with apparently healty cows]

Determination of protein pattern of lymph nodes of 25 cows that had clinical bovine leucosis and comparison with apparently healthy cows. A totalof 25 samples of BLV infected lymph nodes from slaughtered cows that were positive in ELISA and AGID serological tests and had clinical singes of leucosis along with five apparently healthy lymph nodes from cows that were negative in ELISA and AGID for BLV. These tissues were obtained during Jun.2001 to Oct.2002 in Iran. Following protein extraction and purification of the samples electrophoresis by SDS-PAGE method were done on the samples. Protein bands stained by commassie brilliant blue. The results of SDS-PAGE test of samples, were shown that all of the tissues posses 25 different protein bands that related to normal lymph node in addition two 51, 24 kd bands in protein patterns of affected cows. Those two proteins are the most important viral proteins of the BLV and detecting of these proteins can use in diagnosis of the disease

[ study on cellular changes in inoculated cell cultures with Pestivirus, using direct immunoflurescence technique]

Objective: this study was carried out to find the progressive cellular changes developed in cell culture with cytopathic [cp] and noncytopathic [ncp] pestiviruses isolated from cow and sheep

Design: observational study

Procedure: following inoculating the R-BK cell cultures in Lighton tube with NADL strain of BVD, a cp strain of Border disease virus and a nocytopathic BVD, the cells were stained with FA and observed with IF microscope. In inoculated cultures with cp and ncp pestivirus obvious changes were present at the first day and multiple focal expression of viral antigens were seen

Results: in the second day in cp inoculated cultures, the morphological changes began and antigenic spread were more intensive. In the 3rd day the CPE foci were seen in different parts of the cultured cells with high concentration of antigen around the CPE sites. The cells started to detach and became rounds and finally in the 4th and 5th day, almost all cells were antigen positive and developed CPE. However, those cultures inoculated with noncytopattuc strain of BVD, in daily observation the antigenic spread was too slow and up to 6 days post inoculation no CPE were observed