Ecology and genomic features of infection with Mycobacterium avium subspecies paratuberculosis in Egypt.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, or Johne's disease, in cattle, with potential involvement in cases of Crohn's disease in humans. Johne's disease is found worldwide and is economically important for both beef and dairy industries. In an effort to characterize this important infection in Egypt, we analysed the ecological and genomic features of recent isolates of M. paratuberculosis. In this report, we examined 26 Holstein dairy herds distributed throughout Egypt, from 2010 to 2013. Using PCR analysis of faecal samples, we estimated a mean herd-level prevalence of 65.4 %, with animal-level infection that reached a mean of 13.6 % among animals suffering from diarrhoea. Whole genome sequencing of field isolates identified numerous single nucleotide polymorphisms among field isolates relative to the standard M. paratuberculosis K10 genome. Interestingly, the virulence of M. paratuberculosis isolates from Egypt revealed diverse virulence phenotypes in the murine model of paratuberculosis, with significant differences in tissue colonization, particularly during the chronic stage of infection. Overall, our analysis confirmed that Johne's disease is a newly identified problem in Egypt and indicated that M. paratuberculosis has potentially diverse genotypes that impact its virulence. Further ecological mapping and genomic analysis of M. paratuberculosis will enhance our understanding of the transmission and evolutionary dynamics of this pathogen under natural field conditions.

Can Porphyromonas gingivalis be a novel aetiology for recurrent miscarriage?

OBJECTIVE: To study the association between Porphyromonas gingivalis (P. gingivalis) infection and recurrent miscarriage. METHODS: This case control study included women with early pregnancy failure admitted for surgical evacuation of retained products of conception. Cases (group 1) included 50 women with unexplained recurrent early miscarriage whereas the control group (group 2) consisted of 50 women with no such history. The evacuated products of conception, subgingival plaques, cervicovaginal secretions and saliva of all participants were examined to detect P. gingivalis deoxyribonucleic acid (DNA) using a polymerase chain reaction. RESULTS: The prevalence of P. gingivalis DNA in the chorionic villous tissue samples of group 1 was significantly higher than in group 2 (8 [16%] vs. 1 [2%], respectively; p = 0.036, odds ratio [OR]: 9.3, 95% confidence interval [CI]: 1.1-76.9). The prevalence of P. gingivalis DNA was significantly higher in cervicovaginal secretions of group 1 than in group 2 (9 [18%] vs. 1 [2%], respectively; p = 0.02, OR: 10.8, 95% CI: 1.3-88.5). On the contrary, P. gingivalis DNA could not be detected in subgingival plaques and saliva samples of either group. CONCLUSION: The current study found an association between P. gingivalis infection of the female genital tract and the occurrence of recurrent miscarriage.

Molecular diagnosis of acute and chronic infection of Trypanosoma evansi in experimental male and female mice.

Trypanosoma evansi is enzootic in camels in Egypt, and water buffaloes act as a reservoir for camel infection. Molecular techniques have contributed towards understanding the epidemiology of T. evansi. Trypanosoma evansi was detected in acute and chronic stages of the disease in male and female mice by polymerase chain reaction (PCR) using two primers. Two experiments were conducted. In experiment I, two groups consisting of 26 female and 26 male mice received 104 trypanosome by I/P inoculation for each mouse. In experiment II, 42 female and 42 male mice were inoculated I/P with 102 trypanosome/mouse. In addition, five mice were kept as uninfected control for each group. Mice were monitored daily for parasitaemia level during the pre-patent period using the micro-haematocrit centrifugation technique (MHCT) and conventional PCR. The primer pairs, (Trypanosoma brucei) TBR1/2 and TeRoTat1.2 (T. evansi Rode Trypanozoon antigen type [RoTat] 1.2), detected the infection after 24 hours earlier than MHCT in both experiments. The course of infection that was detected by MHCT revealed three waves of parasitaemia in female mice and two waves in male mice in the chronic stage of infection. In addition, PCR was able to detect T. evansi in different organs in the chronic stage (i.e. disappearance of parasite from blood). Application of the two primer sets on blood samples from camels showed that all samples were positive by TBR1/2 primers and only 32 of 44 were positive by TeRoTat1.2 primers. Acutely and chronically Trypanosoma-infected mice were detected by PCR in blood and organs. TBR1/2 primers were more sensitive than TeRoTat1.2 primers in detecting Trypanosoma-infected mice, and more reliable in detecting field-infected camels and excluding carrier animals.

Graphene oxide nanosheets induced genotoxicity and pulmonary injury in mice.

Graphene and graphene-related materials have broadly applied in biomedical purposes due to their unique properties, thus safety evaluation of them is crucial. This study was performed to explore the genotoxic and pulmonary toxic potential of different doses of graphene oxide nanosheets' (GOs) in mice.A total of 90 male mature mice were randomly divided into six groups of fifteen mice per each, five groups were intraperitoneally injected by GO at doses of 10, 50, 100, 250 and 500µg/kg b.w once weekly in addition to the control group that was injected intraperitoneally with 0.2ml saline solution. Five animals from each group were euthanized after 7, 28 and 56days post treatment. Evaluation of genotoxicity was performed through detection of chromosomal aberrations in bone marrow while assessment of lung injury was made by determination of DNA fragmentation in lung specimens using the alkali Comet assay, pulmonary oxidative markers estimation and finally histopathological investigations. Results revealed that GOs induced variable structural chromosomal aberrations (SCA) in bone marrow and DNA damage of lung cells that were time and dose dependent and represented by increase in%DNA in comet tail, tail moment and tail length and decrease in% head DNA in nuclei of lung of GOs-treated mice versus control groups in addition, GOs induced various changes in pulmonary oxidative stress parameters that were affected by dose and duration of treatment compared with the control as well as various pulmonary histopathological alterations were detected indicating lung injury. CONCLUSION: GO potentiate the induction of genotoxicity and pulmonary injury in mice in time and dose dependent manner.

Application of touchdown enzyme time release (TETR)-PCR for diagnosis of Chlamydophila abortus infection.

Chlamydophila abortus-DNA was detected using a touchdown enzyme time-release (TETR)-polymerase chain reaction (PCR) assay as an improved test for sensitive and rapid diagnosis of abortion in small ruminants. Two hundred and fifty two placentae, liver or spleen tissue samples from aborting ewes and goats or aborted lambs and kids in which C. abortus infection was suspected were examined by TETR-PCR and the results were compared with cell culture. Sixty-five tissue samples were found to be TETR-PCR positive while only 56 samples were cell culture-positive. After resolution of discrepant samples with a confirmatory nested PCR assay, TETR-PCR had a sensitivity of 97% and a specificity of 99.5% while culture had a sensitivity of 84.8% and a specificity of 100%. The analytical sensitivity of the TETR-PCR assay was determined with DNA extracted from 4-fold serial dilution of C. abortus B577 culture and found to be 0.25 inclusion-forming unit per PCR. No reduction in the analytical sensitivity was noted when the assay was tested with mouse liver samples spiked with 4-fold serial dilution of C. abortus B577 culture. No target product was amplified when DNA from Chlamydophila pecorum was tested. TETR-PCR used in this study is a practical, rapid, sensitive and specific assay that could be used for the detection of C. abortus in infected tissue samples. We recommend the use of this assay as a supplemental diagnostic tool for detection of C. abortus in infected tissue samples.

Comparison of polymerase chain reaction and cell culture for the detection of Chlamydophila species in the semen of bulls, buffalo-bulls, and rams.

Two hundred and thirty six semen samples were collected from 120 bulls, 60 buffalo-bulls, and 56 rams located on farms of known history of infection with Chlamydophila species. All semen samples were examined by polymerase chain reaction (PCR) and cell culture techniques for detection of Chlamydophila species. The primers were selected to allow the amplification of all target species in a single reaction by identifying conserved sequences in the omp2 gene. PCR assay detected more positive samples (36) from the semen samples collected from different animal species than were detected by the culture method (21). The results indicated that all culture-positive semen samples (21) from different species were PCR positive. The detection limit of the PCR assay was determined with DNA extracted from fourfold serial dilution of C. abortus (B577) and C. pecorum (11/88) cultures and found to be 0.25 inclusion-forming units (IFU) per PCR, while the culture method could not detect less than 4 IFU. This is the first report using PCR for the detection of Chlamydophila species in buffalo-bulls' semen and the assay provides a simple, sensitive, rapid, and reliable means for the detection and identification of the organism.