Application of a Multiplex PCR Assay for Molecular Identification of Pathogenic and Non-Pathogenic Leptospires based on lipL32 and 16S rRNA Genes.

Leptospirosis is a serious zoonotic infection and the most prevalence disease is in the tropical and subtropical region. The definitive diagnosis of Leptospirosis, caused by spirochetes of the genus Leptospira infection is already using culture methods, serological tests such as the microscopic agglutination test (MAT) and molecular detection methods (PCR) are possible. In this study, we used multiplex PCR method for detection of pathogenic and non - pathogenic Leptospira based on lipL32 and 16S rRNA genes. All serovars were obtained from the Leptospira Reference Laboratory of Microbiology Department, Razi Vaccine and Serum Research Institute, Karaj, Iran. The PCR product for the lipL32 and 16S rRNA genes was 272 bp and 240 bp respectively. The sensitivity amplification for the multiplex assay was 10-6 pg / µl for 16S rRNA gene and 10-4 pg / µl for lipL32 gene. The sensitivity for multiplex PCR was 10-3 pg / µl. The results supported the idea that multiplex PCR can be used to detect Leptospira samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much easily than conventional methodologies. Due to the slow growth of Leptospira and the importance of time in diagnosis, molecular methods such as PCR are suggested.

Detection of invA, sivH, and agfA Virulence Genes in Salmonella spp. Isolated from Broiler Breeder Farms in Alborz Province, Iran.

Salmonellosis, among poultry infectious diseases, not only imposes economic losses in the field of poultry breeding but also is considered a zoonotic disease. This study aimed to investigate the presence of invA, sivH, and agfA virulence genes in Salmonella species. The present study was conducted on 30 Salmonella strains. Samples were cultured on selective and differential media, and afterward, the isolates were serotyped using specific antisera based on the Kauffman-White table. Subsequently, the samples were analyzed to detect invA, sivH, and agfA genes by polymerase chain reaction technique. The results indicated that 30 (100%) isolates had invA and agfA virulence genes and 28 (93.33%) isolates had a sivH virulence gene. The highest frequency of serotypes was related to Salmonella infantis. Among the studied serotypes, Salmonella uno and Salmonella O35 lacked the sivH virulence gene, unlike other serotypes. The findings of this study could pave the way for Salmonella monitoring and be used as a pattern to detect Salmonella bacteria-bearing genes encoding invasion and fimbria.

Seroepidemiological Analysis of Leptospiral infection using MAT in Stray Dogs in Alborz, Iran.

Leptospirosis is a zoonotic disease with global importance, and the animals are the source of transmission of this disease through shedding in their urine. Accordingly, it is essential to conduct epidemiological studies of leptospirosis in order to diagnose this disease in dogs and reduce the risk of transmission to humans. This study aimed to perform a seroepidemiological analysis of Leptospiral infection in stray dogs in Alborz, Iran, using the Microscopic Agglutination Test (MAT). In total, 110 blood samples were collected from stray dogs to detect the antibodies against leptospira interrogans serovarsby the MAT. The prevalence rate of positive MAT tests in stray dogs was estimated at 21.84%. The following protocol confirmed that the most common titers were 1:200 (50%) and 1:400 (25%). In addition, the most prevalent Leptospira serovars were L. Canicola (33.33%), and the lowest belonged to L. Pomona (4.1%). Moreover, no significant difference was observed between the age and gender of the dogs regarding their MAT titer (P>0.05). The results also showed a high prevalence of leptospirosis in stray dogs of Koohsar in Alborz province, Iran. Since Leptospirosis is a zoonosis disease, it should be studied continuously in humans and animals, especially dogs.

The effect of cytoplasmic crude extracts of Trichophyton verrucosum on cell mediated immunity.

OBJECTIVE: Trichophyton verrucosum is a slow growing dermatophyte responsible for a number of skin diseases such as ringworm, and is characterized by patches of hair loss and thick crusts on the host skin in domestic animals. In this study, we examined the immunomodulatory effects of crude extract of Trichophyton verrucosum (TV)cytoplasm in a mouse model. METHODS: The TV variate was cultured on Sabouraud dextrose agar and the mycelium was grinded by mechanical force. The purified protein was obtained from crude extract of the fungus, and protein concentration was measured by BradFord assay. Six to eight week-female BALB/c mice were divided into three groups: test group, receiving cytoplasmic crude extract plus defibrinated sheep blood; control group, receiving defibrinated sheep blood; and normal group, receiving normal saline. Injections were performed on days 0, 3, 5, 7 and 9 and the mice were sacrificed four days after the last injection. T lymphocyte metabolic activity was examined by methyl thiazol tetrazolium (MTT) assay, and also interleukin-4 (IL-4) and interferon-γ (IFNγ) levels were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: MTT assay showed that the TV extract stimulated lymphocyte metabolic activity. ELISA results showed that despite increase in the level of IFNγ, no changes were observed in IL-4 level. CONCLUSIONS: Results indicated that crude extract of TV cytoplasm may probably act as an immune modulator, which affects Th1 responses. The TV crude extract may be an appropriate agent to induce cellular immunity for combating dermatophytosis infection in animals; and therefore, TV extract may have some potential applications in vaccine/adjuvant technology.

Comparison of two disc diffusion methods with minimum inhibitory concentration for antimicrobial susceptibility testing of Neisseria gonorrhoeae isolates.

BACKGROUND: A few studies are available comparing either minimum inhibitory concentration (MIC) values with the Clinical and Laboratory Standards Institute (CLSI) disc diffusion method or MIC with the Australian Gonococcal Surveillance Program (AGSP) method. AIM: This study was conducted with the aim to identify the most feasible and cost-effective method for antimicrobial susceptibility testing of Neisseria gonorrhoeae. MATERIALS AND METHODS: Antimicrobial susceptibility testing of N. gonorrhoeae was performed using, in parallel, the E-test for MIC determination and disc diffusion by CLSI and AGSP techniques, and were compared. Susceptibility to penicillin, ciprofloxacin, tetracycline, ceftriaxone and spectinomycin and cefixime were determined by CSLI and AGSP method and Kappa statistics used to analyse the data with SPSS software. RESULTS: All isolates were susceptible to ceftriaxone and spectinomycin by three methods. Ninety-nine (99%) strains were resistant to ciprofloxacin, while 1% showed intermediate susceptibility to ciprofloxacin by all methods. Statistically, there was a moderate level of agreement between the methods for penicillin. CONCLUSION: All three methods gave reproducible results. Although the media used in the disc diffusion by the AGSP method is easy and cheap to prepare, the CLSI method of disc diffusion testing is recommended for susceptibility testing of gonococcal isolates because of its feasibility and 100% accuracy, with MIC by E-test as the reference method.

Alginate nanoparticles as a promising adjuvant and vaccine delivery system.

During last decades, diphtheria has remained as a serious disease that still outbreaks and can occur worldwide. Recently, new vaccine delivery systems have been developed by using the biodegradable and biocompatible polymers such as alginate. Alginate nanoparticles as a carrier with adjuvant and prolong release properties that enhance the immunogenicity of vaccines. In this study diphtheria toxoid loaded nanoparticles were prepared by ionic gelation technique and characterized with respect to size, zeta potential, morphology, encapsulation efficiency, release profile, and immunogenicity. Appropriate parameters (calcium chloride and sodium alginate concentration, homogenization rate and homogenization time) redounded to the formation of suitable nanoparticles with a mean diameter of 70±0.5 nm. The loading studies of the nanoparticles resulted in high loading capacities (>90%) and subsequent release studies showed prolong profile. The stability and antigenicity of toxoid were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ouchterlony test and proved that the encapsulation process did not affect the antigenic integrity and activity. Guinea pigs immunized with the diphtheria toxoid-loaded alginate nanoparticles showed highest humoral immune response than conventional vaccine. It is concluded that, with regard to the desirable properties of nanoparticles and high immunogenicity, alginate nanoparticles could be considered as a new promising vaccine delivery and adjuvant system.

Glanders outbreak at Tehran Zoo, Iran.

BACKGROUND AND OBJECTIVES: In December 2010 four, lions and one tiger died at the Tehran zoo. Out of all samples, Burkholderia mallei (causative agent of Glanders) was isolated just from ulcer sample of the tiger which was imported to Iran from Russia. MATERIALS AND METHODS: One nasal swab from a tiger and fifteen blood samples with anticoagulant belonging to one tiger and fourteen lions (four dead lions and eleven live lions) were collected and were inoculated directly onto the selective media. The isolate was identified by morphological and biochemical and API BBL tests and PCR using specific primers (Bma- IS407-flip). The standard (Razi Type Culture Collection RTCC: 2375) and tiger isolates were inoculated into 2 guinea pigs. All residue solipeds and carnivores were checked by Malleination test and Complement Fixation (CF) Test respectively. RESULTS: One isolate of B. mallei was isolated from tiger's nasal swab. Both of B.mallei strains were recovered from inoculated animals. All of solipeds were negative by malleination test, however, 11 lions including 4 dead and 7 live lions out of 14 lions were positive in CF test for Glanders and all were put down by the authorities. CONCLUSION: Active surveillance of Glanders is essential for solipeds, especially it's more important while being used to feed valuable carnivores like lions and tigers. Therefore, a reliable test like malleination must be carried out twice (first before transferring and one month after quarantine). Both test results should be negative for use for feeding.

Correlation between In vitro susceptibility and treatment outcome with azithromycin in gonorrhoea: a prospective study.

PURPOSE: This prospective study was carried out to determine the antimicrobial susceptibility of Neisseria gonorrhoeae isolates by disc diffusion method and minimum inhibitory concentration (MIC) by E -test with special reference to azithromycin. Also, the correlation between in vitro susceptibility and treatment outcome with single 2 g oral dose azithromycin was assessed. METHODS: The study included 75 gonococcal isolates from males with urethritis, females with endocervicitis and their sexual contacts. All isolates were subjected to susceptibility testing for penicillin, ciprofloxacin, tetracycline, ceftriaxone, spectinomycin, cefixime and azithromycin. Males with gonococcal urethritis were randomised to receive a single dose of either azithromycin or ceftriaxone. Forty-two men with urethritis received 2 g single oral dose azithromycin, while all other patients were given 250 mg parentral ceftriaxone. All patients were called for follow-up to assess clinical and microbiological cure rates. RESULTS: While all the isolates were susceptible to ceftriaxone, spectinomycin, cefixime and azithromycin; 74 (98.7%), 24 (32%) and 23 (30.7%) strains were resistant to ciprofloxacin, penicillin and tetracycline respectively, by both disc diffusion method and E -test. The MIC range, MIC50 and MIC90 of N. gonorrhoeae strains, to azithromycin were 0.016-0.25, 0.064 and 0.19 microg/mL, respectively. Follow-up attendance of the patients was 52.4 with 100% clinical and microbiological cure rates. CONCLUSIONS: Results of our study indicate that 2 g single oral dose azithromycin is safe and effective in the treatment of uncomplicated gonorrhoea.

Epidemilogical analysis of Neisseria gonorrhoeae isolates by antimicrobial susceptibility testing, auxotyping and serotyping.

PURPOSE: This study was carried out to analyze the epidemiology of gonorrhea based on antimicrobial susceptibility testing, auxotyping and serotyping in New Delhi, India. METHODS: Sixty gonococcal isolates from males with urethritis, females with endocervicitis and their sexual contacts were studied. The isolates were subjected to antimicrobial susceptibility testing, auxotyping and serotyping for epidemiological characterization. RESULTS: We observed nine antibiotic resistance patterns. Ninety-eight percent of isolates were resistant to ciprofloxacin, while 20% isolates were penicillinase producing N. gonorrhoeae (PPNG) and 18.3% isolates were tetracycline resistant N. gonorrhoeae (TRNG). Eight auxotypes were observed, of which the NR (non-requiring), proline requiring and arginine requiring were most common auxotypes. On the basis of serotyping alone, the gonococcal isolates could be differentiated into three serogroups and 18 serovars. Serogroup WI represented 46.7% and WII/III represented 51.7% of isolates and one strain was WI and WII/WIII serogroup combination. When results of auxotyping and serotyping were combined (A/S) 29 A/S classes could be identified. The most prevalent A/S classes were NR/Aost, NR/Arost, Pro/Aost and Pro/Boprt. CONCLUSIONS: Although A/S typing had the highest discriminatory index, isolates recovered from index case and their sexual contacts were found to be identical by all typing methods.

Simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis by PCR in genitourinary specimens from men and women attending an STD clinic.

Neisseria gonorrhoeae and Chlamydia trachomatis are the two most common bacterial sexually transmitted infections that manifest primarily as urethritis in males and endocervicitis in females, though the infection may be asymptomatic especially in women. Since complications may occur in untreated symptomatic and asymptomatic infected individuals, early diagnosis and treatment of infected individuals is required to prevent severe sequelae and spread of these diseases. Recently molecular amplification assays like Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) have been found to be highly sensitive and specific methods for detection of N. gonorrhoeae and C. trachonmatis not only in urethral and cervical specimens but also in urine. The objective of this study was to screen male and female Sexually Transmitted Disease (STD) clinic attenders, with and without symptoms suggestive of urethritis and cervicitis for presence of N. gonorrhoeae and C. trachomatis using a multiplex PCR based assay, to compare its performance with culture for N. gonorrhoeae and Direct Fluorescent Antibody (DFA) staining for C. trachomatis and also to compare the efficacy of PCR test performed on urine and genital swab specimens collected from this high risk group. Genital specimens and urine was collected from STD clinic attenders. N. gonorrhoeae and C. trachomatis was detected in genital specimens by culture and DFA respectively. Multiplex PCR was used to detect N. gonorrhoeae and C. trachomatis infection in both genital and urine specimens. Among men with urethritis, N. gonorrhoeae was detected in 70% by culture and 77% by PCR, while C. trachomatis as detected in 7.5% by DFA and 17.5% by PCR. Among females with endocervicitis, N. gonorrhoeae was detected in 7.7% by culture and 30.7% by PCR, while C. trachomatis was detected in 7.7% by DFA and in 15.4% by PCR. None of the asymptomatic males were positive for N. gonorrhoeae and C. trachomatis by conventional methods, while 43.9% were positive for N. gonorrhoeae and 7.5% for C. trachomatis by PCR. Fifty per cent of asymptomatic women were positive for C. trachomatis by PCR alone. We encountered PCR positive but culture/DFA negative results and also PCR negative but culture/DFA positive results. In view of this a single PCR test cannot be used for diagnosis and treatment of N. gonorrhoeae and C. trachomatis infection unless confirmed by a second test.

Blocking adherence of uropathogenic Escherichia coli isolate to HEP-2 cells and bladder of mice in the presence of antibody against p-fimbriae.

Presence of fimbriae on Escherichia coli isolated from the urine of patients with urinary tract infection was related to the ability of the bacteria to attach to human uroepithelial cells. One of the 50 isolates that expresses high MRHA p-fimbriae, selected and antibody against p-fimbriae from it, showed blocking of attachment of bacteria to HEP-2 cell in 1:1024 titer. Also, 1:512 titer of this antiserum to blocking of attachment in bladder tissue of mice is significant.