Archives of Razi Institute
Volume:66 Issue: 2, Autumn 2011

During 2009-10, real time RT-PCR and conventional RT-PCR techniques Used for detecting BTVs RNA in 310 blood samples. For real time and gel based RT-PCR segment-1 and segment-10 selected as conserve genes to search any BTV strains. Using these methods, 58 (%18.7) and 14 (%4.5) positive samples were detected among the clinically suspected sheep. Sensitivity of both molecular techniques evaluated by log-10 serial dilutions of BTV16 RNA, and determined 101.8 and 103.8 TCID50/ml in rRT-PCR and conventional RT-PCR respectively. This report confirmed rRT-PCR assay could detect weak BTV positive samples even at end stage of infection. In this study Virus isolation from selected positive samples failed by inoculation to embryonated chicken egg, Vero and KC cell.

In this research a molecular method based on polymerase chain reaction for typing of Clostridium perfringens was developed and toxin genotypes of 64 isolates from sheep and goats in Iran were determined. The PCR assays were developed for detection of alpha (cpa), beta (cpb) and epsilon (etx) toxin genes, allowing classification of the isolates into genotypes A B, C and D. The field isolates were assigned to genotypes A (n=9, 14.07 %), B (n=20, 31.25%), C (n=17, 26.56%) and D (n=18, 28.12%). In this PCR system the fragments of 900, 611 and 402 bp were amplified using specific primers for alpha, beta and epsilon toxins, respectively. The fragments were confirmed by sequencing and blasting in GenBank. The sequence alignment of the fragments showed more than 98% similarity with other related published sequences from other sources. Our results suggest that PCR genotyping is an acceptable tool for in vitro typing of C. perfringens.

Mycoplasma synoviae (M. synoviae) is a major worldwide poultry pathogen that causes serious economic losses in the poultry industry. This study was designed to detect M. synoviae through culture isolation and polymerase chain reaction (PCR) assay to demonstrated the involvement of M. synoviae infection in trachea and the lung/air sac samples taken from commercial broiler chicken farms in 3 main provinces of Iran (Tehran, Markazi and Qazvin), with clinical signs of the disease. Total of 43 samples were cultured in PPLO broth media supplemented for M. synoviae isolation. The bacteria DNAs were extracted by phenol/chloroform method and the PCR assay amplifying the conserved region of 16S rRNA gene was applied for the detection of Mycoplasma genus in 163bp fragment and M. synoviae in 207bp fragment from culture as same as in clinical samples. Of the 43 swabs 28(65.1%) yielded one of the potentially pathogenic mycoplasmas evaluated for using PPLO agar culture diagnostic method, and 33(76.8%) yielded one of the potentially pathogenic Mycoplasmas evaluated for using Mycoplasma genus PCR as diagnostic method, and 24(55.9%) of the swabs yielded M. synoviae for using M. synoviae PCR as diagnostic method. In this study we had observed the highest quantity of M. synoviae infections in broiler chicken with PCR test. In conclusion, PCR is a more rapid, effective, sensitive and inexpensive method than the standard culture technique, that could be used as an alternative method for traditional culture and showed the real number of the M. synoviae contaminated broiler chicken farms.

Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of John’s disease also called paratuberculosis. This is economically one of the important infectious diseases in cattle and ruminant husbandry. This disease is manifested as granulomatosis entrocolitis, lymphadenitis and inflammation local lymphatic vessels. The typical sign of this disease is progressive loss of weight. Considering the importance of detection of this disease in this study, two methods, culture and PCR, were used for the identification of this microorganism. In this study 100 milk samples from apparently healthy cows and 100 milk samples from cows that have been suspicious of John’s disease were taken from in Sarab, East Azarbaijan, Iran. Direct microscope observation after ziehl-neelsen staining was done. Then, bacterial culture on specific medium was carried out, and finally, identification of Mycobacterium avium subsp. paratuberculosis was examined using PCR and specific primers. Using direct observation, culture and PCR analyses showed that from 100 healthy cow milk samples, 8, 9 and 12 samples were positive MAP for each method respectively. The results of direct observation, culture and PCR analysis on affected cows were 15, 40 and 44, respectively. The results of this study showed that culture and PCR analyses methods are important in the identification of the causes of this disease. Therefore, considering the frequency of the disease in the studied region, either of those methods can be used in the microorganism identification.

American foulbrood (AFB) is the most serious brood disease of the honey bee. Traditional methods are reliable but rather slow simply because they are based on biochemical, morphological and physiological identification of cultivated isolates. The aim of this study is the detection of Paenibacillus larvae larvae spores in honey and diseased larvae samples by culture and PCR. Therefore 54 samples of diseased larvae and 36 honey samples, were diluted with an equal volume of distilled water and centrifuged, then the pellet was used for bacterial culture, DNA extraction and PCR. PCR products were electrophoresed on 0.8 % agarose gel. Five of 54 (9.3 %) larvae samples and 5 of 36 (13.9 %) honey samples were positive for Paenibacillus larvae larvae by culture and PCR. As a result, screening of honey and larvae samples by PCR method proved to be a reliable, fast and useful method on regional and national scale for monitoring, controlling, and using preventive measures before the occurrence of American foulbrood damages.

Marek's disease (MD) is a common lymphoproliferative disease of chickens, usually characterized by mononuclear cellular infiltration in different organs. The disease is caused by a herpes virus and is transmissible. MD has been a common, important problem for poultry industry worldwide, as well as, in our country, Iran. The aim of present study was to have an estimation of the incidence of MD in broiler flocks of some major regions of chicken rearing in Tehran province (Savojbolagh, Karaj, Shahriar and Varamin). This was implemented by 35 times visiting of some poultry slaughterhouses and thoroughly inspection of chicken's carcasses and histopathological examination of various tissues and organs of suspected and normal slaughtered chickens from 80 broiler flocks, that was reared in Tehran province. Gross and microscopic examinations of chickens, in four mentioned regions, showed that 24 out of 80 flocks (30%) had been infected to different forms of the disease. This result indicated that MD has a high incidence in broiler flocks of Tehran province. The incidence of cutaneous, visceral and mixed cutaneous and visceral forms in these regions (four regions) was determined as 16.2%, 3.8% and 10%, respectively. Moreover, no case of nervous and ocular forms was seen in this study. The result of the current study gives a hint for the importance and losses behind the high incidence of the MD in broiler flocks of Tehran province, Iran. Further detailed study on MD in broiler flocks and on the effectiveness of available MD vaccines in reducing the incidence and losses of the disease is recommended.

Thirty isolates of Bacillus anthracis recovered from animal carcasses, soil and human in different localities in Iran between 2007 and 2008. They were tested by standard disc diffusion susceptibility method for their resistant/ susceptibility to different kinds of antibiotics. According to American National Committee of Clinical Laboratory Standards (NCCLS) guidelines all isolates were sensitive to levofloxacin (100%), cefixime (100%). Other isolates showed different kinds of sensitivity to: doxycycline (96.7%), cephalothin (95.6%), ampicillin (95.6%), nitrofurantoin (95.6%), tetracycline (94.4%), ofloxacin (89.9%), gentamycin (77.8%), nalidixic acid (72.2%), kanamycin (75.6%), erythromycin (71.1%), piperacillin (78.9%), tobramycin(64.4%), choramphenicol (59.9%), cefotaxime (33.3%), ciprofloxacin (1.1%), cefuroxime (33.3%), azithromycin (44.4%), streptomycin (55.6%), ticacillin (35.6%), rifampicin (34.4%), clindamycin (74.4%), ceftriaxon (26.7%), methicillin (55.6%), trimethoprim (8.9%), cloxacillin (31.1%) and penicillin (74.4%). One of the isolates was complete resistance to penicillin.Therefore, preventive and therapeutic strategies involving the use of antibiotics should take the possibility of resistance and/or susceptibility of the isolates into account and not decided without antibiotic sensitivity testing.

Ornithobacterium rhinotracheale (ORT) is a respiratory pathogen which has been isolated throughout the world from numerous bird species. The present study was designed to isolate and characterize the ORT from domestic turkeys, quails and pigeons. For this purpose, 250 samples from each bird species (turkey, quail and pigeon) with or without respiratory signs were tested by taking of tracheal swabs. In addition, respiratory tissue samples (tracheal and lung), from 250 slaughtered turkeys, 50 slaughtered quails and 100 dead pigeons were also subjected to culture for ORT as tracheal swabs. Respiratory tissues were also tested for bacterial DNA by using polymerase chain reaction (PCR). In general, 30 isolates including 4 isolates from turkeys, 3 isolates from quails and 23 isolates from pigeons were identified as ORT by bacteriological method and then confirmed by PCR. Bacterial DNA was detected in 20%, 50% and 35% of respiratory tissues in turkeys, quails and pigeons respectively. Five ORT isolates from pigeon and all four isolates from turkey showed smaller colony size, while other isolates had larger colonies when cultured in blood agar. Fifty percent of the isolates with larger colony but none of the isolates with small colony size could agglutinate red blood cells (RBCs). All of the isolates were sensitive to danofloxacin and chloramphenicol while more than 90% of pigeon isolates were resistant to ampicillin. All of turkey and quail and 30% of pigeon isolates were resistant to tetracycline. Our ORT isolates showed high identity (98%- 100%) in sequence of 16S rRNA gene to related data in GeneBank.

A survey was carried out to investigate the frequency of hard tick species (Acari: Ixodidae) on sheep in Khorasan Razvi province. A total of 812 ticks were collected from the sheep of different areas of Khorsan Razavi province five species were identified as follow: Rhipicephalus turanicus (59.23%), Hyalomma.marginatum turanicum (25.73%), Hyalomma excavatum (14.8%), Hyalomma anatolicum (8.3%), and Dermacentor niveus (4.8%). The frequency of tick infestation in southern parts was greater than northern parts of the province. R. turanicuss and H. m. turanicum. Were dominant ticks in the province.

In tropical and subtropical countries, envenomation by scorpions (so-called scorpionism) represents a serious public health problem. In the present study, the toxic effects of mice LD50 injections of Mesobuthus eupeus (Me) venom on the kidney and liver of anesthetized rabbits were investigated. Six rabbits were selected and ALT, AST, BUN and creatinine were measured at 0, 1 and 3 hours after envenomation and histopathological studies were carried out postmortem. All the animals showed signs and symptoms of envenomation within 30-40 minutes and died 3 to 3.5 hours after venom injection. Histopathological examinations revealed glumerolar congestion, dilated vessels of interstitium and focal interstitial congestion in the kidney and focal hemorrhage, central vein congestion, congested vessels in portal areas and dilated sinusoids in the liver at 3 to 3.5 hrs following venom injection. In addition, biochemical analyses indicated significant rise in the levels of ALT and creatinine following Mesobuthus eupeus envenomation in animals at 3 hrs. However no significant changes were observed at 1 hr. In conclusion, scorpion (Mesobuthus eupeus) venom leads to damage in vital organs such as liver and kidney.

Hemiscorpius lepturus is the most dangerous scorpion species, endemic in Khuzestan province and other southwestern areas of Iran, causing morbidity and mortality in children and adults. The efficacy of the antivenom for reversal of manifestation caused by venom of this scorpion is a controversial issue. In the present work, H. lepturus venom (1500 µg/kg) was subcutaneously injected into two separate groups of rabbits. Groups 1 and 2 of rabbits received antivenom through i. v route, at 1 and 3 hours after venom injection respectively. Electrocardiograms of all the rabbits recorded during the experiment. In group 1 rabbits blood collection carried out, before and 1 hr after venom injection as well as 3 and 24 hours after antivenom injection. In group2 animals, blood collection was carried out before and 3 hours after venom injection as well as 3 and 24 hours after antivenom injection. Separated serum used for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), creatine kinase isoenzyme, MB (CK-MB), urea، creatinine and Blood Urea Nitrogen (BUN). In group 1 rabbits highly significant (P<0. 01) increase in level of CPK, CK-MB and significant (P<0. 05) increase in level of urea, creatinine and BUN within 3 and 24 hours after antivenom injection was observed. In group 2, highly significant (P<0. 01) elevated level of CPK, CK-MB and significant (P<0. 05) elevated level of urea and creatinine within 3 hours after antivenom injection was observed. Moreover, highly significant (P<0. 01) elevated level of CPK, CKMB and AST and significant (P<0. 05) elevated level of rest of the biochemical parameters within 24 hours after antivenom injection was observed. On the other hand, in these groups, electrocardiogram abnormalities such as ST elevation and sinus bradycardia in limb lead II, were indicator of a mild cardiac injury. In conclusion, results obtained in this study indicate that the efficacy of antivenom for reversal of cytotoxic manifestations occurring after envenomation is time limited. Therefore, it is recommended to use antivenom as early as possible before the occurrence of serious cytotoxic effect on various organs.

This syndrome is a shock-like hyper metabolic myopathy triggered in susceptible animals by stress. Capture myopathy (C.M.) is a commonly occurring condition in mammals following trapping and transportation. In this case 12 to 24 hours after transportation of red deer (Cevus elaphus) and wild goats (Capra ibex) clinical signs such as: muscular tremor, ataxia, recumbency, hyperthermia, tachycardia, hyperventilation and red brown urine observed. According to symptoms Capture myoparthy was diagnosed Treatment was ineffective on one red deer and one wild goat. Necropsy findings of dead animals were included: hyperemia, petechial hemorrhage in pericardium and heart muscle, pale foci of leg and heart muscles and red brown urine in bladder. This case report represents the attention to Capture myopathy in wild animals and particular caution that should be exercised in capturing and handling of these animals.