Comparison of three methods including temperature, H2O2/ascorbic acid/sonication, and nitrous acid treatments for overcoming the inhibitory effect of heparin on DNA amplification in realtime-PCR.

Heparin molecules have an inhibitory effect on DNA amplification by binding to the majority of DNA-interacting proteins. Different physical, chemical, and enzymatic methods have been used to degrade and depolymerize heparins in biomedical investigations. In this study, we aimed to evaluate some heparin degradation methods to eliminate the inhibitory effect of heparin on DNA amplification. Here, we report highly efficient, simple, and convenient methods to eliminate the heparin inhibitory effect on DNA amplification by treatments including temperature, nitrous acid, and H2O2/ascorbic acid/sonication. Further, treatment conditions including temperature degree and duration of treatments, the concentration of ascorbic acid, and intensity of sonication were reviewed. Target DNAs were extracted using the phenol-chloroform method. DNA concentrations and purity were analyzed before and after each treatment by Nanodrop spectrophotometry. DNA amplifications were attempted using a commercially available realtime-PCR mastermix. We found that the inhibitory behavior of heparin was well eliminated after the 85 °C/2 h, 65 °C/2 h, nitrous acid (pH = 3), and H2O2/ascorbic acid/sonication treatments, respectively. The further analyses indicated that the application of nitrous acid in pH = 1.5 and H2O2/ascorbic acid/sonication in higher ascorbic acid concentrations and sonication intensities lead to failure in DNA amplification due to the degradation of target sequences. From our experience, simple heat treatments or at the next level using nitrous acid and H2O2/ascorbic acid/sonication have enabled the detection and quantification of virus infection in heparin blood samples. These approaches may enable researchers to utilize blood taken in heparin tubes for genome amplification and diagnostic purposes.

Genetic characterization and phylogenetic of Anaplasma capra in Persian onagers (Equus hemionus onager).

Anaplasma spp. are among the most recognized arthropod-borne infectious agents. Although the novel A. capra has been isolated from wildlife, livestock, and hard ticks from many parts of the world, there is no report regarding the identification of this pathogen from equines and little is known about the epidemiology of A. capra in Equidae. In this study, A. capra was identified in two out of ten blood specimens of wild onagers (Equus hemionus onager) during a routine health check-up in Semnan, Iran by light microscopy and molecular analyses while other pathogens were not detected. First, inclusions on RBC's were observed in two blood smears by light microscopy. Then, the blood specimens of both animals were analyzed by realtime-PCR for Anaplasma, Ehrlichia, and Theileria infections. A 1400 bp sequence of 16S rRNA belonging to Anaplasmataceae and 874 bp fragment for groEL gene for A. capra were amplified in Anaplasma positive samples and sequenced. Preliminary BLAST analysis of sequenced fragments showed high homology to A. capra strains in GenBank database. Finally, nested PCR and restriction enzyme fragment length polymorphism techniques confirmed the pathogen as A. capra. To the best of our knowledge, this study has reported the occurrence of A. capra in wild onagers for the first time and suggests that equines could be infected with this pathogen and act as reservoirs for A. capra.

The First Study of West Nile Virus in Feral Pigeons (Columba livia domestica) Using Conventional Reverse Transcriptase PCR in Semnan and Khorasane-Razavi Provinces, Northeast of Iran.

BACKGROUND: West Nile Virus (WNV) is an arboviral infection continuing to be as major threat to human health as well as the livestock industry all around the world. Birds including pigeons are one of the potential reservoirs for WNV. This study aimed to detect the presence of WNV genome in feral pigeons circulating in Semnan and Khorasane-Razavi Provinces (Iran) including 10 urban and 10 suburban areas. METHODS: Totally, 150 samples (brain and kidney) were collected equally from feral pigeons and the presence of WNV genome was evaluated in these samples after RNA extraction. RESULTS: All the samples were negative for the presence of WNV-RNA in this investigation. CONCLUSION: Although obtained result indicated no evidence of WNV genome in feral pigeons but complementary studies regarding serologic detection of WNV in vertebrate hosts as well as pigeons and identification of arthropod vectors seems necessary for comprehensive determination about infection status in these areas.

Comparison of Cell-Free Fetal DNA Plasma Content Used to Sex Determination Between Three Trimesters of Pregnancy in Torkaman Pregnant Mare.

This investigation aimed to compare the cell-free fetal DNA (cffDNA) plasma present in three trimesters of pregnancy in Torkaman pregnant mare. Peripheral blood samples of 32 pregnant mares in three trimesters of pregnancy were collected in tubes containing ethylenediaminetetraacetic acid at three time points. Circulating cffDNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, a conventional polymerase chain reaction was performed for the sex-determining region Y (SRY) gene present in the Y chromosome. Of the total 32 Torkaman pregnant mares, 24 were carrying male fetuses and eight were carrying female fetuses. In total, the accuracy of the test was 48.75%, 68.75%, and 75% in the first, second, and third trimesters of pregnancy, respectively. The sensitivities were 25%, 58.32%, and 66.66%, respectively, whereas their specificities were 100% in all trimesters. In conclusion, the SRY gene can permit the detection of equine fetal sex with good accuracy through cffDNA analysis in maternal plasma just in the third trimester of pregnancy, although specificity in all duration of pregnancy was 100%.

Co-administration of Erythromycin and Leech Salivary Extract Alleviates Osteomyelitis in Rats Induced by Methicillin-Resistant Staphylococcus aureus.

OBJECTIVE: Erythromycin (Ery) and leech saliva (LS) can inhibit Staphylococcus aureus growth in in vitro conditions. This study aimed to evaluate the activities and synergy between Ery and LS on chronic osteomyelitis in male Wistar rat's tibia induced by methicillin-resistant S. aureus (MRSA). MATERIALS AND METHODS: Four weeks after osteomyelitis induction, rats were divided into four groups including no treatment (control), Ery monotherapy (orally), LS monotherapy, or Ery + LS twice daily for 2 weeks. Staphylococcus aureus growth, pathological signs and inflammatory cytokine tumour necrosis factor-alpha (TNF-α) levels were assessed. RESULTS: Rats tolerated all therapeutic strategies well during the experiment. The Ery treatment alone significantly decreased bacterial growth, pathological signs and TNF-α levels. Leech saliva alone reduced TNF-α level significantly, but did not produce a significant reduction in bacterial growth and pathological signs. Ery + LS treatment significantly decreased bacterial growth, considerably alleviated bone pathological signs and decreased TNF-α levels compared with other groups. Statistical analysis suggested that there was a stronger efficiency and synergistic action of Ery and LS when combined against MRSA-induced osteomyelitis in rats. CLINICAL SIGNIFICANCE: The present study suggests that LS may have clinical utility to treat MRSA-induced osteomyelitis when combined with Ery or other therapeutics.

Comparative virulotyping and phylogenomics of Escherichia coli isolates from urine samples of men and women suffering urinary tract infections.

OBJECTIVES: Escherichia coli strains are common pathogens that can cause urinary tract infections (UTI). This study aimed to assess E. coli phylogroups and virulence types in male and female UTI patients. MATERIALS AND METHODS: In the present study, 160 uropathogenic E. coli (UPEC) isolates (from both sexes) were assigned to phylogroups/types and some extraintestinal virulence factors were detected within them by multiplex-PCR. RESULTS: The isolates from women and men were predominantly distributed within phylogroup B2 and D, respectively. The presence of D2 phylotype was higher in men isolates than women, significantly (P=0.045). In male isolates papEF and sfa/focDE are more prevalent in B2 group than D, significantly (P=0.048; P=0.035). The prevalence of hly in B2 group is significantly higher than D (P=0.034) in female isolates. CONCLUSION: This study highlighted different features of E. coli genotypes from phylogenetic and virulence point of view implicated in UTI's in both human genders.

Comparative restriction enzyme mapping of Campylobacter jejuni isolates from turkeys and broilers based on flaA flagellar gene using HpyF3I endonuclease.

Turkeys and broilers have been identified as important reservoirs for Campylobacter jejuni which is of public health significance. The evaluation of the genotypes among C. jejuni strains within different reservoirs is critical for our understanding of the epidemiology of this infectious agent. The present study aimed to compare the genetic diversity and differences of C. jejuni isolates from turkeys and broilers using flagellin PCR-RFLP typing (flaA typing) technique, in terms of the ease of use and discriminatory power. Sixty C. jejuni isolates were detected biochemically and confirmed by duplex-PCR from turkeys and broilers (30 strains from each bird species). Then, a flaA gene fragment (1725 bp) of C. jejuni isolates was amplified and amplicons were digested with HpyF3I enzyme. Restriction analysis by HpyF3I gave four different flaA patterns (H1, H2, H3, H4) among all tested C. jejuni isolates. In broiler isolates, all four patterns were observed but in turkey isolates, only H2 and H4 patterns were present. The results clearly demonstrated that distribution of the flaA typing patterns differed depending on the host species (broiler/turkey). H1 and H3 flaA types are more prevalent in broiler than turkey isolates, while H2 type is significantly more prevalent within isolates from turkey (p < 0.05). The flaA typing technique by digestion with HpyF3I enzyme can almost give us a clue to the source of infection in local outbreaks.

Prevalence of cerebral toxoplasmosis among slaughtered sheep in Semnan, Iran

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii. Felids are definitive hosts and all warm-blooded animals can be intermediate hosts. Some animals such as sheep, goats and pigs are sensitive to infection. In sheep production systems, toxoplamosis can cause abortion and economic loss. In public health, this disease can be transmitted to humans by the consumption of undercooked infected meat or other organs. In this study, T. gondii DNA was detected by B1 gene amplification in 140 randomly-selected brains of slaughtered sheep in Semnan, Iran. The prevalence of ovine cerebral toxoplasmosis was estimated using 95% confidence interval. The brain was selected as a target organ because it gives the highest detection rates, and the results can be compared with previous data from other countries. Our findings indicate that T. gondii is present in ovine tissues and can be passed on to humans by consuming undercooked or raw meat and other organs such as the liver. The infection can be lethal for immunosuppressed individuals and can cause abortion or birth of infected children in pregnant woman.

The Role of TNF-α in Aflatoxin B-1 Induced Hepatic Toxicity in Isolated Perfused Rat Liver Model.

Aflatoxin B-1 (AFB1) is one of the major mycotoxins causing food contamination. Previous studies have shown that AFB1 can induce carcinogenicity and toxic effects in the isolated perfused rat liver and these effects are associated with its metabolites and peroxidation activity. Here we surveyed whether these pathogenic effects of AFB1 are associated with TNF-α as an inflammatory cytokine in general liver damages. In this study, we used twenty male Wistar rats (250-300 g). Rats were divided into four groups. Control group was pre-treated with LPS and then perfused with KHBB. The second group was pretreated with PTX and LPS and then perfused with KHB. The third group was pre-treated with LPS and then perfused with AFB-1 and KHB. The last group was pretreated with LPS and PTX and then perfused with AFB1 and KHB. Results revealed that aflatoxin B1 significantly increased the enzyme activity of aminotransferase and levels of lipid peroxidation. Also, the levels of Glutathione decreased in the aflatoxin group significantly. TNF-α released in perfusate and increased in aflatoxin B1 group significantly and decreased in AFB-1+PTX. Exposure to Aflatoxin B1 may induce reactive oxygen species, so these species may induce overproduction of proinflammatory cytokines such as TNF-α and may cause more damage to hepatic cells.

Pathotyping of diarrhoeagenic cattle Escherichia coli strains isolated in the Province of Tehran, Iran.

An oligonucleotide DNA microarray targeting 348 virulence factors and genetic markers was used in the pathotyping, serotyping and phylogrouping of 51 Escherichia coli strains isolated from faecal samples. The samples were collected from diarrhoeic 1 to 30 days old calves located at 14 farms in the Tehran province, Iran. Positive microarray signals for genes encoding the Locus of Enterocyte E acement (LEE), the Type III Secretion System (TTSS), and the absence of EPEC adherence factor (EAF) permitted the pathotyping of 25 strains as atypical Enteropathogenic (aEPEC) or Enterohaemorrhagic Escherichia coli (EHEC). The lack of LEE and TTSS-associated genes distinguished the remaining 26 strains, which were classi ed as Extraintestinal pathogenic E. coli (ExPEC). Atypical EPEC belonged to phylogroup B1 and possessed a LEE pro le tir-1, eae(beta), espA-1, espB-3. The EHEC strains primarily belonged to the B1 phylogroup type-O26 and possessed either a LEE pro le tir-1, eae(beta), espA-1, espB-3, or a B1 type-O111, LEE tir-3, eae(gamma), espA-1, espB-2. ExPEC-typed strains generally harboured genes localised to the constant region of Colicin V plasmid (pColV), including increased serum survival factor (iss), complement resistance protein (traT), aerobactin operon (iucD), and the siderophore receptor (iroN). The microarray platform used in this study is well suited to accurately and rapidly type attaching and e acing E. coli (AEEC-types), thus providing a database for the meta-analysis of ExPEC-typed strains.

Distribution of Shiga toxin genes subtypes in B1 phylotypes of Escherichia coli isolated from calves suffering from diarrhea in Tehran suburb using DNA oligonucleotide arrays.

BACKGROUND AND OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) have emerged as human pathogens and contamination via animal origin has been a major public health concern. We compared the distribution of phylogenetic groups and prevalence of stx gene variants among the pathogenic strains of Escherichia coli isolated from feces of diarrheatic calves in Tehran suburb farms. MATERIALS AND METHODS: In this study we screened 140 diarrheatic calves (1-15 days old) for E. coli strains during a 3 months period of time. The isolated strains were grouped into different phylotypes according to the presence of chuA, yjaA and TSPE4.C2 genes. Then, the prevalence of stx gene subtypes was evaluated in the B1 phylotypes. RESULTS: From diarrheatic calves, 51 bacterial isolates were biochemically identified as E. coli and 31 isolates out of 51 were considered B1 phylotype using DNA Microarray technology. Of these isolates, 20 contained stx1a and stx1b and one harbored all mentioned variants of stx genes except stx2b2 . CONCLUSION: This study showed that in Tehran suburb, the B1 phylotype of E. coli is prevalent as a causative agent of diarrhea in calves and the prevalence of stx1 gene subtypes is dominant in comparison with other subtypes. Considering the possibility that these stx genes can be spread to other strains, bovine E. coli strains are an important source of stx genes for other strains and further study and surveillance seems to be required for the exact identification of virulence profile of E. coli phylotypes in different hosts.

Genetic characterization of Escherichia coli O157:H7 strains isolated from the one-humped camel (Camelus dromedarius) by using microarray DNA technology.

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.