Thirumahal Vanniasinkam

Adenoviral Gene Delivery for HIV-1 Vaccination

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the proteins structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

Genetic characterization of equine adenovirus type 1

Two known serotypes of equine adenovirus (EAdV), equine adenovirus type 1 (EAdV-1) and equine adenovirus type 2 (EAdV-2) have been isolated from horses. EAdV-1 is predominantly associated with upper respiratory tract infections while EAdV-2 appears to be associated with gastrointestinal infections in horses. In this report the EAdV-1 genome has been sequenced for the first time. The EAdV-1 genome encoded genes are characteristic of the Mastadenovirus genus such as protein V and IX. Unexpectedly, phylogenetic reconstructions also revealed a close relationship between EAdV-1 and two recently characterized bat adenoviruses. The results of this study suggest that EAdV-1 may share a common ancestor with canine and bat adenoviruses.

Prevalence of equine adenovirus antibodies in horses in New South Wales, Australia.

There are currently two known serotypes of equine adenovirus (EAdV), equine adenovirus type 1 (EAdV1) and equine adenovirus type 2 (EAdV2); EAdV1 is predominantly associated with upper respiratory tract infections while EAdV2 appears to have a higher association with gastrointestinal infection, however, very little is known about the prevalence of these viruses in horse populations in Australia. In this study we tested 122 serum samples obtained from horses in New South Wales, Australia, using a standard serum neutralization (SN) assay and ELISA. Ninety-seven of the 122 sera displayed had moderate to high titers of antibodies to EAdV1 and/or EAdV2. Eighteen of the 122 sera were positive for both EAdV1 and EAdV2. In contrast, only thirty-seven positive samples were detected using the ELISA. These results suggest that EAdV1 and EAdV2 infections are widely prevalent in the horse population tested and that SN is currently the most suitable assay for the detection of EAdV1 and EAdV2 antibodies in equine serum.

Rhodococcus equi (Prescottella equi) vaccines; the future of vaccine development

For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular-based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.

Determining the stability of complete blood count parameters in stored blood samples using the SYSMEX XE-5000 automated haematology analyser

In this study, changes that occur in various blood parameters as determined by the Sysmex XE‐5000 analyser upon storage of blood samples for 72 h were investigated.

Prevalence of Shiga toxin-producing Escherichia coli (STEC) in Tasmania, Australia.

Aims: (1) To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and the incidence of STEC associated enteritis within Tasmania and thereby evaluate the need for routine testing for this organism, and (2) compare detection method(s) for this purpose. Methods: 282 selected faecal samples were tested for STEC using selective culture media and multiplex tandem PCR (MT-PCR). The PCR used detected the virulence markers stx1, stx2 and eaeA. Results: STEC were isolated in six of 282 faecal samples. This represents a STEC notification rate for Tasmania of 2.3 cases per 100u200a000 population. CT-SMAC medium detected one, CHROMagar STEC medium detected three and MT-PCR detected six STEC isolates. CHROMagar STEC medium displayed 50% sensitivity and 93% specificity compared to MT-PCR. Conclusions: The incidence of STEC associated enteritis in Tasmania was almost six times greater than the previously reported Australian average. STEC Screening Easy-Plex MT-PCR was more sensitive than both CHROMagar STEC and CT-SMAC media for the detection of STEC from pre-enriched faecal samples.

Plant Phenols as Antibiotic Boosters: In Vitro Interaction of Olive Leaf Phenols with Ampicillin

The antimicrobial properties of olive leaf extract (OLE) have been well recognized in the Mediterranean traditional medicine. Few studies have investigated the antimicrobial properties of OLE. In this preliminary study, commercial OLE and its major phenolic secondary metabolites were evaluated in vitro for their antimicrobial activities against Escherichia coli and Staphylococcus aureus, both individually and in combination with ampicillin. Besides luteolin 7‐O‐glucoside, OLE and its major phenolic secondary metabolites were effective against both bacteria, with more activity on S. aureus. In combination with ampicillin, OLE, caffeic acid, verbascoside and oleuropein showed additive effects. Synergistic interaction was observed between ampicillin and hydroxytyrosol. The phenolic composition of OLE and the stability of olive phenols in assay medium were also investigated. While OLE and its phenolic secondary metabolites may not be potent enough as stand‐alone antimicrobials, their abilities to boost the activity of co‐administered antibiotics constitute an imperative future research area. Copyright

Characterisation of the Equine adenovirus 2 genome

Equine adenovirus 2 (EAdV-2) is one of two serotypes of adenoviruses known to infect equines. Initial studies did not associate EAdV-2 infections with any specific clinical syndromes, although more recent evidence suggests that EAdV-2 may be associated with clinical and subclinical gastrointestinal infections of foals and adults respectively. In contrast, Equine adenovirus 1 is well recognised as a pathogen associated with upper respiratory tract infections of horses. In this study the complete genome sequence of EAdV-2 is reported. As expected, genes common to the adenoviruses were identified. Phylogenetic reconstructions using selected EAdV-2 genes confirmed the classification of this virus within the Mastadenovirus genus, and supported the hypothesis that EAdV-2 and EAdV-1 have evolved from separate lineages within the adenoviruses. One spliced open reading frame was identified that encoded for a polypeptide with high similarity to the pIX and E1b_55K adenovirus homologues and was designated pIX_E1b_55K. In addition to this fused version of E1b_55K, a separate E1b_55K encoding gene was also identified. These polypeptides do not appear to have evolved from a gene duplication event as the fused and unfused E1b_55K were most similar to E1b_55K homologues from the Atadenovirus and Mastadenovirus genera respectively. The results of this study suggest that EAdV-2 has an unusual evolutionary history that warrants further investigation.

Isolation of Dermatophytes (and Other Fungi) from Human Nail and Skin Dust Produced by Podiatric Medical Treatments in Australia

BACKGROUNDnPodiatric physicians routinely use electric drills for the treatment of nail and skin conditions. The grinding process produces human nail and skin dust that is generally vacuumed into bags in the grinding unit. Many of the nails are thought to be mycotic, particularly because they are obtained from patients with symptoms of dermatophyte infections. Currently, there is limited information available on the detection of fungi from nail dust samples. Herein, we attempt to address this situation and outline some of the difficulties that pathology laboratories face in isolating and identifying dermatophytes from nail samples.nnnMETHODSnFifty nail dust bags from podiatric medical clinics across all of the states and territories of Australia were collected and analyzed. Samples from the bags were inoculated onto primary isolation media. Fungal colonies that grew were then inoculated onto potato dextrose agar for identification using standard morphological (macroscopic and microscopic) features.nnnRESULTSnOne hundred fifty-one colonies of dermatophytes were identified from 43 of the 50 samples. In addition 471 nondermatophyte molds were isolated, along with some yeasts and bacteria.nnnCONCLUSIONSnThe most common dermatophytes isolated were from the Trichophyton mentagrophytes/interdigitale complexes. Trichophyton rubrum, Trichophyton tonsurans, Trichophyton soudanense, and Epidermophyton floccosum were also isolated. An unidentified group of dermatophytes was also present. The three most common genera of nondermatophyte molds were Aspergillus, Penicillium, and Scopulariopsis, all of which have been implicated in onychomycosis and more general disease. The presence of viable fungal pathogens in the dust could potentially pose a health problem to podiatric physicians.