Production of the phospholipase D and heat-shock protein [HSP]-60 recombinant proteins from Corynebacterium pseudotuberculosis

Background: Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable losses for herd owners. Phospholipase D [PLD] is a potent exotoxin produced by C. pseudotuberculosis and it has been considered as the major virulence factor for this bacterium, possibly contributing to the spread of the bacteria from the initial site of infection to secondary sites within the host. Heat shock proteins [HSPs] are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune re-sponses in mammals

Objectives: The aim of this study was the cloning and expression of the PLD and HSP genes of C. pseudotuberculosis

Methods: PLD and HSP[60] genes were cloned into pMAL-c2X vector and recombinant plasmids construct was transformed to DH[5] strain of E. coll. Expression of the proteins was shown by SDS-PAGE and accuracy of the cloned genes was confirmed by nucleo-tide sequence analysis

Results: The transformed E. coll strain DH[5] expressed PLD and HSP60 proteins effectively. The expressed fusion protein was found almost entirely in the soluble form

Conclusions: In the following studies the immunogenicity and protectivity of these recombinant proteins against C. pseudotuberculosis infections can be assessed

[Comparison of antibacterial effects of nanoparticles of magnesium oxide and silicon oxide on methicillin resistant bacteria]

.Background and Aim: Considering bacterial resistance to common antibiotics and the need for new drugs, use of medicinal products manufactured by nano-technology, can be effective in the prevention and treatment of bacterial infections. In this study, we evaluated the sensitivity of Staphylococcus aureus and Salmonella to nanoparticles of magnesium oxide and silicon oxide in vitro

Methods: Staphylococcus intermedius and Methicillin-resistant Staphylococcus aureus and Salmonella typhimurium, Salmonella abourtus were cultured in Mueller Hinton Broth medium. Then different concentrations of the nanoparticles of magnesium oxide and silicon oxide were added to the culture medium. After 24 hours of incubation we measured optical density [OD] by means of ELISA reader. Multi-well plate was used as controls. Using SPSS software data were analyzed by ANOVA and LSD post hock test

Results: Silicon nanoparticles prevented growth of Staphylococcus intermedious, Staphylococcus aureus, Salmonella typhimurium and Salmonella abortus [p<0.0001] in a dose dependent way, but showed no antibacterial effect on Salmonella typhi. Also nanoparticles of magnesium oxide showed antimicrobial effect on the above-mentioned bacteria in a dose-dependent manner [p<0.000l]

Conclusion: Silicon oxide and magnesium oxide nanoparticles can be used and evaluated as antibacterial drugs in experimental or clinical infections

[ serological survey on strangles disease in horses of some areas in Khuzestan province by ELISA]

Background: strangles is caused by Streptococcus equi subspecies equi. The bacteria typically infect the upper respiratory system and lymph nodes of the head and neck in equidae

Objectives: the aim of this study was to evaluate the prevalence of strangles and association of this infection with host age and geographical determinants in horses in Khuzestan province

Methods: serum samples from 184 horses were randomly collected in Ahvaz, Shoushtar, Baghmalek, Shoush, Abadan, Ramhormoz and Dezfool cities and were examined by ELISA assay. Also, 85 swab samples were randomly taken from nasal swab of horse and evaluated for Streptococcus equi subspecies equi by bacterial culture

Results: seroprevalence rate of strangle was 37.5% [95% CI: 30.5-44.5%]. Logistic regression mshowed that the odds of infection between the age based on year and disease was 1.1 [95% mCI: 1.04-1.17] [p<0.001], and with increase of 1 year-old, odds of infection increase 10%. Relative frequency of infection in male and female horses was 32.73 and 39.53%, respectively [p>0.05] and odds of infection in female compared with male horses was 1.34 [95% CI: 0.69-2.61]. Prevalence rate in horses with and without history of respiratory disease was 94.1% and 31.74%, respectively [p<0.001]. The odds of infection in horses with history of respiratory disease compared with healthy horses was 34.42 [95%CI: 4.45-266.37]. Prevalence rate in Ahvaz, Shoushtar, Baghmalek, Shoush, Abadan, Ramhormoz and Dezfool was 33.3%, 34.62%, 5.26%, 69.23%, 13.04, 22.22% and 75%, respectively [p<0.001]. Geographical location explained 29.6% of infection's fluctuations. No isolate of Streptococcus equi subsp. equi was obtained in culture of nasal swab samples

Conclusions: this study determined that seroprevalence of strangles should be deleted in khuzestan province is high and Prevention and control measurements should be considered by health authorities

Evaluation of immunopathologic effects of aqueous extract of Echinacea purpurea in mice after experimental challenge with Pasteurella multocida serotype A

In order to assess the immunopathological effects of aqueous Echinacea purpurea extract [EPE] on mice experimentally challenged with Pasteurella multocida serotype A, forty female BALB/c mice were randomly divided into four groups. The groups included a control group [received sterile distilled water 2 times/week for 2 weeks, intraperitoneally and then 100 microl sterile saline intranasally], a PMA group [received sterile distilled water as the control group and after 2 weeks, 5.6 x 10[3] CFU/ml of P. multocida serotype A, intranasally], an EPE+PMA group [received E. purpurea extract intraperitoneally 2 times/week for 2 weeks and then challenged as the PMA group] and an EPE group [received E. purpurea extract as EPE+PMA group and then 100 microl sterile saline intranasally]. After 24 and 48 h post challenge, half of the animals in each group were sacrificed and analyzed for bacterial counts in their lungs and livers, TNF[alpha] serum levels and histapathological changes. The results showed significant differences in lung bacterial counts between PMA and EPE+PMA groups. TNF[alpha] serum level was significantly higher in the PMA group. Histopathological examination revealed infiltration of neutrophils in alveolar septa and hyperemia in the PMA group. In addition, the criteria of bronchopneumonia were partially recovered in the EPE+PMA compared to the PMA group. According to the results, it seems that E. purpurea extract has an immunomodulatory effect and can be used to prevent or control of pneumonia caused by Pasteurella

[Association between amino acid sequences in peptide binding region [BOLA-DRB3] and susceptibility or resistance to calf diarrhea]

The major histocompatibility complex genes [mhc] encode MHC I and II molecules which present peptide fragments to T cells. Therefore these polymorphic molecules critically influence susceptibility to infectious diseases. At present study potential relationship between amino acid sequences in the antigen binding groove of different BoLA-DRB3 alleles and susceptibility or resistance to calf diarrhea was investigated. Twelve different DRB3 alleles were found among 171 calves [84 diarrheic and 87 healthy] analyesd by PCR-RFLP method. Amino acid sequences of the encoded peptide binding region were compared. 26 polymorphic positions were detected in this region. A significant association [p<0.05] was shown between occurrence of diarrhea and the presence of glutamic acid and tyrosine inpocket 4 and valine, glutamine and leucine in pocket 9 of peptide binding region. Thus it can be concluded that pockets 4 and 9 of the BoLA-DRB3 molecule would be involved in conferring susceptibility of calf to diarrhea

[Cloning, sequencing and expression of HSP70 c-terminal domain in Mycobacterium avium subsp. paratuberculosis]

Heat shock proteins [HSPs] have been shown to act as an adjuvant when co-administered with different antigens, especially tumor antigens or antigens from infectious agents. C-terminal domain of Mycobacterium tuberculosis heat shock protein 70 [Hsp70], when fused to peptide antigens, provides a unique structure that is able to induce potent immune responses. In this study, aneukaryotic expression vector pEGFP-N1, containing C-terminal domain of Mycobacterium paratuberculosis HSP 70, Green Fluorescent Protein [GFP] gene in the plasmid construct, was designed for use as a reporter. With GFP system, expression of the target protein was evaluated in the cell culture. The nucleotide sequence of the cloned gene was revealed by sequencing. The protein expression of designed plasmid was also proved by reverse transcriptase polymerase chain reaction [RT-PCR]. Our eukaryotic expression vector [pEGFP-N1 -hsp70 c-terminal] was successfully constructed and HSP70 c-terminal domain protein was expressed effectively. The current experiment, as a basis for a new DNAvaccine design, can be used for the future studies on reverse vaccinology