Michael Kotiw

Late-onset and Recurrent Neonatal Group B Streptococcal Disease Associated with Breast-milk Transmission

The purpose of the study was to determine the epidemiological relationships in three unrelated cases of neonatal late-onset Group B streptococcal (GBS) disease and maternal breast-milk infection with GBS. All deliveries were by cesarean section; case 1 was at term, and cases 2 and 3 were at 32- and 33-wk gestation, respectively. Case 1 relates to a mother with clinical mastitis and recurrent GBS infection in a 20-day-old male infant. Following antibiotic therapy and cessation of breastfeeding, the infant recovered without sequelae. Case 2 refers to a mother with clinical mastitis and the occurrence of late-onset GBS disease in 5-wk-old male twins. Despite intervention, one infant died and the second became ill. Following antibiotic therapy and cessation of breast-feeding, the surviving infant recovered without sequelae. Case 3 refers to a mother with sub-clinical mastitis and late-onset GBS infection occurring in a 6-day-old female twin. Following intervention, the infant recovered but suffered a bilateral thalamic infarction resulting in developmental delay and a severe seizure disorder. Following recovery of GBS from an inapparent mastitis and cessation of breast-feeding, the second infant remained well. Blood cultures from all affected infants and maternal breast milk were positive for GBS. Epidemiological relationships between neonatal- and maternal-derived GBS isolates were confirmed by a random amplified polymorphic DNA polymerase chain reaction assay (RAPD-PCR). This study is significant in that it has demonstrated that maternal milk (in cases of either clinical or sub-clinical mastitis) can be a potential source of infection resulting in either late-onset or recurrent neonatal GBS disease.

Differentiation of Infectious Laryngotracheitis Virus Strains Using Restriction Endonucleases

Strains of infectious laryngotracheitis virus (ILTV) were examined using an indirect immunofluorescent test (IIF) and with restriction endonucleases for detecting intratypic differences. Electrophoretic analysis of ILTV DNA fragments cleaved with restriction endonuclease Hind 111 clearly distinguished between strains. The IIF test did not discriminate between strains. A molecular weight estimate of ILTV DNA was made by summation of restriction endonuclease fragments cleaved with BamH1 (102.1 X 10(6)) and Hind111 (97.35 X 10(6)). Differences between the estimates may indicate the presence of submolar fragments.

The effect of serial in vivo passage on the expression of virulence and DNA stability of an infectious laryngotracheitis virus strain of low virulence

The effect of 35 serial passages in vivo on an infectious laryngotracheitis virus strain of low virulence was examined in terms of effect on virulence and DNA stability. Within 3 passages in live chickens there was evidence of increasing respiratory distress. Severe respiratory distress (with death in some cases) was observed after the 6th passage, except when there appeared to be a transient decline in pathogenicity following short term storage of the virus inoculum at -70 degrees C. Restriction endonuclease analysis of viral DNA derived from the original inoculum and the final passage did not reveal any genomic alteration. It is postulated that there is a potential for live ILTV vaccines to cause outbreaks of clinical disease in the event of inadequate or incomplete vaccination procedures.

Isochromophilone IX,a Novel GABA-Containing Metabolite Isolated from a Cultured Fungus Penicillium sp.

Isochromophilone IX, a novel antibiotic active component was isolated from a penicillium species and investigated. Isochromophilone IX displays a specific rotation with the same sign as isochromophilone VI. It was found that isochromophilone represents the first occurrence of an γ-amino butyric acid (GABA) containing metabolite among only four naturally occuring members of this structure class.

Caelestines A-D, brominated quinolinecarboxylic acids from the Australian ascidian Aplidium caelestis.

Four new brominated natural products, caelestines A-D (1-4), have been isolated from the Australian ascidian Aplidium caelestis. The structures of 1-4 were determined by analysis of their NMR and MS data. This is the first report of brominated quinolinecarboxylic acids from nature. Compound 1 has been previously synthesized but not spectroscopically characterized. Compounds 1-4 were tested against three mammalian cell lines (MCF-7, NFF, and MM96L) and a panel of microbial strains and showed only minor cytotoxicity.

Immunological response to parenteral vaccination with recombinant hepatitis B virus surface antigen virus-like particles expressing Helicobacter pylori KatA epitopes in a murine H. pylori challenge model

ABSTRACT Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S–KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 103) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 102). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 104 and 2.6 × 104, respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.

Restriction endonuclease analysis of group B streptococcal isolates from two distinct geographical regions

This study examined isolates of group B streptococci (GBS) by latex seroagglutination and restriction endonuclease analysis (REA). REA analysis was performed with high- and low-frequency cutting enzymes, and fragments were resolved by linear and pulsed-field gel electrophoresis (PFGE), respectively. GBS isolates were derived from a regional acute-care hospital and a tertiary referral obstetric hospital. The most common serotype isolated at the general hospital was serotype V (42%). A high incidence of this serotype V was associated with wound infections, but there was no predominant REA profile. Serotype III was the most common from the obstetric hospital (41%). The majority of these isolates were associated with neonatal GBS infection and there was a predominant restriction pattern. Analysis of restriction patterns of GBS isolated from individual mother/ baby pairs revealed common patterns. These data extend the evidence for vertical transmission of GBS and clones of serotype III GBS with increased pathogenic potential.

Differentiation between virulent and avirulent strains of infectious laryngotracheitis virus by DNA:DNA hybridization using a cloned DNA marker.

The DNAs of virulent and avirulent strains of infectious laryngotracheitis virus (ILTV) showed greater than 96% homology by reciprocal DNA:DNA hybridization. Nevertheless, by use of selected restricted DNA fragments, it was possible to differentiate strains according to pathotype more readily than by the more laborious restriction enzyme analysis. Restricted DNA fragments were successfully cloned into Escherichia coli HB101 cells and could be used not only for pathotyping ILTV strains but also for their differentiation from other avian viruses.

Anti-Inflammatory Activity and Structure-Activity Relationships of Brominated Indoles from a Marine Mollusc

Marine molluscs are rich in biologically active natural products that provide new potential sources of anti-inflammatory agents. Here we used bioassay guided fractionation of extracts from the muricid Dicathais orbita to identify brominated indoles with anti-inflammatory activity, based on the inhibition of nitric oxide (NO) and tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages and prostaglandin E2 (PGE2) in calcium ionophore-stimulated 3T3 ccl-92 fibroblasts. Muricid brominated indoles were then compared to a range of synthetic indoles to determine structure-activity relationships. Both hypobranchial gland and egg extracts inhibited the production of NO significantly with IC50 of 30.8 and 40 μg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα and PGE2 with IC50 of 43.03 µg/mL and 34.24 µg/mL, respectively. The purified mono-brominated indole and isatin compounds showed significant inhibitory activity against NO, TNFα, and PGE2, and were more active than dimer indoles and non-brominated isatin. The position of the bromine atom on the isatin benzene ring significantly affected the activity, with 5Br > 6Br > 7Br. The mode of action for the active hypobranchial gland extract, 6-bromoindole, and 6-bromoisatin was further tested by the assessment of the translocation of nuclear factor kappa B (NFκB) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NFκB in the LPS-stimulated RAW264.7 macrophages by 48.2%, whereas 40 µg/mL of 6-bromoindole and 6-bromoistain caused a 60.7% and 63.7% reduction in NFκB, respectively. These results identify simple brominated indoles as useful anti-inflammatory drug leads and support the development of extracts from the Australian muricid D. orbita, as a new potential natural remedy for the treatment of inflammation.

Biological methods: a novel course in undergraduate biology

We describe a novel course in first year undergraduate practical biology, which introduces students to the principles and practice of a variety of biological techniques. In addition, students develop conceptual skills in experimental design, problem solving, gathering and analysis of data and report writing. The course provides a theoretical foundation and repeated practice of a range of laboratory tasks. Students proceed to higher levels of study with experience in the application and use of basic spectrophotometry and light microscopy, the estimation of unknowns from standard curves, volumetric work including the performance of serial dilutions, and gram stains and the maintenance of bacterial cultures. The unit content can be modified to suit specific curriculums without loss of efficiency or impact.