ARCHIVES OF RAZI INSTITUTE
Year:2011 | Volume:66 | Issue:1|Papers Count:13

Designing and producing a proper fusion construction is the most important problem of producing large quantities of a properly folded functional protein. This construction should have all necessary components of a real gene. A good designed fusion gene construction could be cloned into a good and suitable host.Clostridium perfringens is an important pathogen of humans and livestock and produces numerous toxins including epsilon and beta which are responsible for severe diseases. In the present study a new construction containing Clostridium perfringens type D epsilon toxin gene and type B beta toxin gene was designed. At the first step two pairs of primers were used for these genes amplification. At the next step epsilon forward and beta reveres primers were used to produce a chimeric gene containing amplified partial cds of etxD and partial cds of cpbB which are linked together by the AEAAAKEAAAKA fragment as a small linker. The method was based on fusion PCR and using of Pfu DNA polymerase, which has a proofreading activity. The fusion gene inserted into pJET1.2blunt and cloned into E. coli strain TOP10. Based on the latest information, this is the first design and cloning of epsilon-beta fusion gene and also this is the first time that PCR fusion strategy is used for Clostriadial gene fusion, which could be used for development of a recombinant epsilon-beta fusion protein vaccine. This construction also could serve as a model for development and production of novel fusion protein for other potential proteins and toxins.

Contagious agalactiae (C.A.) is one of the most common disease affecting small ruminants which is caused by Mycoplasma agalactiae. This disease is particularly widespread around the world and Iran is one of the countries that C.A. is present. The aim of this study was isolation and identification of M. agalactiae (MG) with culture and PCR technique in milk samples in Kordestan province, Iran. A total of 367 milk samples were collected from sheep and goat. Specific published primers amplify a 375 bp gene of MG were used for PCR. Twenty (5.5%) out of 367 were positive in PPLO agar and 5 (25%) out of these isolates were positive with Mycoplasma agalactiae primers. Four (75%) out of 5 isolates was from sheep and 1 (25%) from goat. Result of PCR with 367 milk samples showed that 11 (3%) of them were positive with these primers. The isolation of M. agalactiae showed that C.A is present in Kordestan province and our results suggested that PCR method because of reduces the time consuming could be an alternative method beside culture.

The measurement of antibody responses of animals exposed to BVDV either through a natural exposure or an immunization protocol is still a standard procedure. For BVDV, the test formats have been largely limited to ELISA which is a valuable diagnostic test to measure the level of BVDV specific antibodies as well as antigen in blood samples. In the present study, 120 blood samples were collected from the cows with the history of abortion in different period of pregnancy from different industrial dairy cattle herds of Mashhad area of Iran. Also 30 samples were collected from the cows with no history of abortion as control. The presence of antibody against BVDV from the 120 serum samples was investigated by indirect ELISA. From 120 serum samples which were collected from aborted cows, 89 samples were positive (%74.16). From these positive samples, 12 (13.48%), 54 (60.68%) and 23 (25.84%) samples belong to the first, second and third trimester of pregnancy, respectively. From 89 positive samples, 12 (13.48%) samples were related to stillbirth and 8 (8.99%) samples were belongs to the mummified fetus. From 89 positive samples, 71 (79.78%) were related to cattle between 2-5 years old and 18 (20.22%) were associated to cattle more than 5 years old. In control group, 20 samples (66.66%) were antibody positive. Also the presence of BVDV antigen in serum samples was investigated by Ag-capture ELISA. From 120 serum samples, 2 samples were positive (1.67%), which belongs to the second period of pregnancy. In control group, none of the samples were antigen positive. The results of this study showed that the prevalence of BVDV infection is high among the aborted cows of Mashhad area. Although this prevalence is higher than the control group, the observed difference is not significant.

In order to estimate the mean of maternal antibody in calves against BHV-1 (Bovine Herpes virus type 1), this study was carried out in a population of calves from non-vaccinated dairy cattle at 2 livestock in Qazvin province. One hundred thirteen sera out of 512 were collected from 1-4 months unvaccinated calves. We used Blocking –Percentage of maternal antibodies against BHV-1, which obtained by Blocking ELISA assay in 1-4 months calves sera. The result of one way analysis of variance determined that there was a significant difference among blocking percentage of maternal antibodies against BHV-1 in 1-4 months (P<0.001). Comparing percentage of mean titer indicated a decreasing trend with respect to age i.e. from 84.4 (a 95% CI: 78.1- 90.6) in 1 month to 57.6 (a 95% CI: 47.1- 68.2) in 4 months, which was near to 55 as cut off point. Tukey's method showed a significant difference between the Blocking percent of mean titer between 1 and 4 (P<0.001), 2 and 4 (P=0.034) months. Chi-square test for independency showed a significant association between age and seroactivity (P=0.005).

Shiga toxin producing Escherichia coli have been associated with HUS, HC and TTP in human. We found recto-anal mucosal sample in sheep as well in cattle is the main site for E. coli O157 localization.1246 E. coli isolates from 872 both healthy and diarrheic animals were analyzed, by screening for the presence of Shiga toxin-producing (VT 1 and VT 2) and intimin (eae) genes used Multiplex PCR.87 (9.75%) VTEC from 52 cattle and 28 (7.90%) from 28 sheep were isolated. VT2 gene was found to be more frequent than VT1 in cattle (54.02% vs 26.43%), in contrast the same genes in sheep (21.42%vs25%). There was observed significant difference in the origin of VT positive sheep in close contact with farms of cattle origin. Having cattle and sheep with each other was a possible risk factor. The animal was kept in pen was more localized than tethered. Young cattle were documented strongly significant high prevalence rate in E. coli O157: H7 than older, but no effect of age was observed on the occurrence of E. coli O157 in sheep. Both diarrheic and healthy animals were shed E. coli O157: H7 in their feces. Sheep and dairy cow were not illustrated any significance differences geographical region and seasonal variation with E. coli O157: H7 prevalence rate.

Pasteurellosis is one of the important economic diseases in ruminants, especially in cows and buffaloes. It is caused by Pasteurella multocida and occasionally by Mannheimia haemolytica. The aim of this study was to isolate Pasteurella multocida from lungs with probable mentioned bacterial agents in cows and buffaloes in Urmia's slaughter house.240 lung samples over a period of 12 months were cultured. The results have revealed 6 (2.5%) Pasteurella multocida results suggest that the animal, its breed, sex, age and season can be effective in the occurrence of these positive cases. The positive samples were all from male beef cattle of hybrid breeds (4 samples) in winter and Holstein breeds (2 samples) in spring.

Heamophilus influenza type-b (Hib) is a gram - negative pleomorphic bacterium that causes meningitis infections in children with the age of less than 5 years particularly in two years old infants. In the present study various isolates of Heamophilus influenzae from infants suspected to meningitis were collected, identified, characterized and were used throughout our experiments. Different culture media namely Brain Heart Infusion Broth (BHIB), Tryptic Soy Broth (TSB) and GC medium Base (GCB) which this medium was modified and prepared in our own laboratory, were compared to determine the highest bacterial yield. All media were added supplements 10mg/ml hemin & 0.01/ml IsovitaleX containing V factor. The bacterial yield for all Hib strains present in our laboratory were measured with an initial inoculums of 104 cfu per ml. The result showed very closed amount of biomass for all isolates however, GCB had slightly higher yield and ultimately we chose this medium for cultivation and extraction of capsular polysaccharide (CPS-b). In our laboratory we have adapted the PRP production according to our technical and instrumental availabilities which exists in our laboratories replacing ultra centrifugation to phenol chloroform to remove contaminants like endotoxin and proteins to the minimum level and also decreased the number of some chemical treatments while some steps were added in purification process. Our study showed although there were not significant differences between the PRP extract of the three isolates with average amount of 108 mg/lit, however, isolate ATCC10210 (ATF2) showed the highest amount with 192 mg/lit and the least PRP was produced by isolate H.inf.1, with 16 mg/lit. It seems that the data can be categorized to a normal distribution with the mean of 106.4 and standard deviation of 6.25. This result was confirmed by one sample kolmogorov-Smirnov test, hence the PRP³192 mg/lit is statistically significant at a significant level of a=0.10 (P=0.085). The amount of contaminants e.g protein along with nucleic acid present in PRP was estimated at optical density 280 and 260nm, were under 10% and 5% respectively.

Lactococcus garvieaeis the etiological agent of lactococcosis, an emerging disease which affects several fish species and causes important economic losses both in marine and freshwater aquaculture. Lactococcosis usually happens when water temperature increases over 15oC during the year. Normally, it causes a hyperacute and haemorrhagic septicemia in fish. This paper presents a procedure for producing experimental vaccine for rainbow trout (Oncorhynchus mykiss) lactococcosis including aspects such as pathogen characterization, pathogenicity, mass cultivation, safety, potency and field trial tests for immersion use. In the potency test, after challenging the vaccinated fish with live pathogenic bacteria (1×107 bacteria per milliliter of immersing solution) and observing for 72 hours thereafter, 10% of fish died while the control group showed 60% mortality within the observation time. In the field trial from vaccination time onward till marketing of the fish, those mortalities that occurred in groups of vaccinated and non-vaccinated fish were recorded. Total death occurred in the vaccinated group was 11%, while in non vaccinated group this number was approaching 23%. This observation indicates a 50% reduction in mortality in the vaccinated group. This is the first report on experimental vaccine against lactococcosis in fish that is produced and tested in Iran.

GenusLeishmania is a protozoan parasite that causes different severe diseases. Fetal Calf Serum (FCS) is the major part of the Leishmania culture media, for mass cultivation and the most expensive ingredient in these media. In the present work, the efficacy of chicken serum was evaluated in Leishmania culture media. The results indicated that, the (10%) chicken serum enriched culture medium supported the growth of large scale cultures of the parasites and can be used for primary and mass cultivation of Leishmania parasites. In conclusion, the chicken serum was effective, easy available and cheap replacement for FCS.