Diphtheria in Lao PDR: Insufficient Coverage or Ineffective Vaccine?

Background

During late 2012 and early 2013 several outbreaks of diphthe-ria were notified in the North of the Lao People’s Democratic Republic. The aim of this study was to determine whether the re-emergence of this vaccine-preventable disease was due to insufficient vaccination coverage or reduction of vaccine effectiveness within the affected regions.

Methods

A serosurvey was conducted in the Huaphan Province on a cluster sampling of 132 children aged 12–59 months. Serum samples, socio-demographic data, nutri-tional status and vaccination history were collected when available. Anti-diphtheria and anti-tetanus IgG antibody levels were measured by ELISA.

Results

Overall, 63.6% of participants had detectable diphtheria antibodies and 71.2% tetanus antibodies. Factors independently associated with non-vaccination against diphtheria were the distance from the health centre (OR: 6.35 [95% CI: 1.4–28.8], p = 0.01), the Lao Theung ethnicity (OR: 12.2 [95% CI:1,74–85, 4], p = 0.01) and the lack of advice on vac-cination given at birth (OR: 9.8 [95% CI: 1.5–63.8], (p = 0.01) while the level of maternal edu-cation was a protective factor (OR: 0.08 [95% CI: 0.008–0.81], p = 0.03). Most respondents claimed financial difficulties as the main reason for non-vaccination. Out of 55 children whose vaccination certificates stated that they were given all 3 doses of diphtheria-containing vaccine, 83.6% had diphtheria antibodies and 92.7% had tetanus antibodies. Furthermore, despite a high prevalence of stunted and underweight children (53% and 25.8%, respectively), the low levels of anti-diphtheria antibodies were not correlated to the nutritional status.

Conclusions

Our data highlight a significant deficit in both the vaccination coverage and diphtheria vaccine effectiveness within the Huaphan Province. Technical defi-ciencies in the methods of storage and distribution of vaccines as well as unreliability of vac-cination cards are discussed. Several hypotheses are advanced to explain such a decline in immunity against diphtheria and recommendations are provided to prevent future outbreaks.     

Analysis of Multiple Brachyspira hyodysenteriae Genomes Confirms That the Species Is Relatively Conserved but Has Potentially Important Strain Variation

The intestinal spirochete Brachyspira hyodysenteriae is an important pathogen in swine, causing mucohemorrhagic colitis in a disease known as swine dysentery. Based on the detection of significant linkage disequilibrium in multilocus sequence data, the species is considered to be clonal. An analysis of the genome sequence of Western Australian B. hyodysenteriae strain WA1 has been published, and in the current study 19 further strains from countries around the world were sequenced with Illumina technology. The genomes were assembled and aligned to over 97.5% of the reference WA1 genome at a percentage sequence identity better than 80%. Strain regions not aligned to the reference ranged between 0.2 and 2.5%. Clustering of the strain genes found on average 2,354 (88%) core genes, 255 (8.6%) ancillary genes and 77 (2.9%) unique genes per strain. Depending on the strain the proportion of genes with 100% sequence identity to WA1 ranged from 85% to 20%. The result is a global comparative genomic analysis of B. hyodysenteriae genomes revealing potential differential phenotypic markers for numerous strains. Despite the differences found, the genomes were less varied than those of the related pathogenic species Brachyspira pilosicoli, and the analysis supports the clonal nature of the species. From this study, a public genome resource has been created that will serve as a repository for further genetic and phenotypic studies of these important porcine bacteria. This is the first intra-species B. hyodysenteriae comparative genomic analysis.     

Characterisation of the nitrile hydratase gene clusters of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> strains AJ270 and AJ300 and <Emphasis Type="Italic">Microbacterium</Emphasis> sp. AJ115 indicates horizontal gene transfer and reveals an insertion of IS1166

The nitrile metabolising strains AJ270, AJ300 and AJ115 were isolated from the same location. The strains have very similar nitrile metabolising profiles. Sequencing of the 16S rRNA gene indicates that strains AJ270 and AJ300 are novel strains of Rhodococcus erythropolis while strain AJ115 is a novel Microbacterium strain very closely related to Microbacterium oxydans and Microbacterium liquefaciens. Analysis of the structure of the nitrile hydratase/amidase gene clusters in the three strains indicates that this region is identical in these strains and that this structure is different to other nitrile hydratase/amidase gene clusters. The major difference seen is the insertion of a complete copy of the insertion sequence IS1166 in the nhr2 gene. This copy of IS1166 generates a 10 bp direct duplication at the point of insertion and has one ORF encoding a protein of 434 amino acids, with 98% homology to the transposase of IS666 from Mycobacterium avium. A gene oxd, encoding aldoxime dehydratase is found upstream of the nitrile hydratase gene cluster and an open reading frame encoding a protein with homology to GlnQ type ABC transporters is found downstream of the nitrile hydratase/amidase genes. The identity of the nitrile hydratase/amidase gene clusters in the three strains suggests horizontal gene transfer of this region. Analysis of the strains for both linear and circular plasmids indicates that both are present in the strains but hybridisation studies indicate that the nitrile hydratase/amidase gene cluster is chromosomally located. The nitrile hydratase/amidase enzymes of strain AJ270 are inducible with acetonitrile or acetamide. Interestingly although a number of Fe-type nitrile hydratases have been shown to be photosensitive, the enzyme from strain AJ270 is not.     

Environmental controls of temporal and spatial variability in CO2 and CH4 fluxes in a neotropical peatland

Tropical peatlands play an important role in the global storage and cycling of carbon (C) but information on carbon dioxide (CO₂) and methane (CH₄) fluxes from these systems is sparse, particularly in the Neotropics. We quantified short and long‐term temporal and small scale spatial variation in CO₂ and CH₄ fluxes from three contrasting vegetation communities in a domed ombrotrophic peatland in Panama. There was significant variation in CO₂ fluxes among vegetation communities in the order Campnosperma panamensis > Raphia taedigera > Cyperus. There was no consistent variation among sites and no discernible seasonal pattern of CH₄ flux despite the considerable range of values recorded (e.g. ?1.0 to 12.6 mg m^?2 h^?1 in 2007). CO₂ fluxes varied seasonally in 2007, being greatest in drier periods (300–400 mg m^?2 h^?1) and lowest during the wet period (60–132 mg m^?2 h^?1) while very high emissions were found during the 2009 wet period, suggesting that peak CO₂ fluxes may occur following both low and high rainfall. In contrast, only weak relationships between CH₄ flux and rainfall (positive at the C. panamensis site) and solar radiation (negative at the C. panamensis and Cyperus sites) was found. CO₂ fluxes showed a diurnal pattern across sites and at the Cyperus sp. site CO₂ and CH₄ fluxes were positively correlated. The amount of dissolved carbon and nutrients were strong predictors of small scale within‐site variability in gas release but the effect was site‐specific. We conclude that (i) temporal variability in CO₂ was greater than variation among vegetation communities; (ii) rainfall may be a good predictor of CO₂ emissions from tropical peatlands but temporal variation in CH₄ does not follow seasonal rainfall patterns; and (iii) diurnal variation in CO₂ fluxes across different vegetation communities can be described by a Fourier model.     

Construction of Recombinant HVT Expressing PmpD,and Immunological Evaluation against Chlamydia psittaci and Marek’s Disease Virus

Chlamydia psittaci (C. psittaci) is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT) is an efficacious commercially available vaccine against Marek’s Disease virus (MDV). In this study, a recombinant HVT-delivered vaccine against C. psittaci and Marek’s disease was developed and examined. The 5''-terminus of pmpD gene (pmpD-N) encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The recombinant virus (rHVT-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-N or parental HVT were protected completely against challenge with a very virulent strain of Marek’s Disease virus (MDV) RB-1B. Post challenge with C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and C. psittaci.     

Localization and induction in early development of Xenopus

Our experimental results, as well as those of others, lead us to suggest the following steps in the dorsalization and axialization of the Xenopus egg and embryo: the sperm aster determines the direction of rotation of the cortex relative to the deeper cytoplasm (endoplasm); the rotation of the cortex activates latent dorsalizing-axializing agents in the vegetal hemisphere. The extent of rotation determines the amount of activation. The direction of rotation determines the location of the activated agents. The activated agents determine the level of mesoderm-inducing activity of the vegetal cells cleaved from that cytoplasmic region. The level of inducing activity determines at least the time at which marginal zone cells will begin gastrulation movements. The time of its initiation of gastrulation may determine how anterior and dorsal a particular marginal zone cell can become.     

Validation of an isocratic HPLC method to detect 2-fluoro-β-alanine for the analysis of dihydropyrimidine dehydrogenase activity

The efficacy of the chemotherapeutic drug 5′-fluorouracil is reduced by catabolism to 2′-fluoro-β-alanine (FBAL), a three-step reaction in which dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step. To study in vitro DPD activity, we developed and validated an isocratic, reverse-phase HPLC method to detect and quantify FBAL without using multiple columns or radiolabeled substrates. Pre-column derivatization of FBAL was performed using o-phthalaldehyde in the presence of two sulfur donors, ethanthiol or β-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10–200 μg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.     

Divergent long‐term trends and interannual variation in ecosystem resource use efficiencies of a southern boreal old black spruce forest 1999–2017

Long‐term trends in ecosystem resource use efficiencies (RUEs) and their controlling factors are key pieces of information for understanding how an ecosystem responds to climate change. We used continuous eddy covariance and microclimate data over the period 1999–2017 from a 120‐year‐old black spruce stand in central Saskatchewan, Canada, to assess interannual variability, long‐term trends, and key controlling factors of gross ecosystem production (GEP) and the RUEs of carbon (CUE = net primary production [NPP]/GEP), light (LUE = GEP/absorbed photosynthetic radiation [APAR]), and water (WUE = GEP/evapotranspiration [E]). At this site, annual GEP has shown an increasing trend over the 19 years (p < 0.01), which may be attributed to rising atmospheric CO₂ concentration. Interannual variability in GEP, aside from its increasing trend, was most strongly related to spring temperatures. Associated with the significant increase in annual GEP were relatively small changes in NPP, APAR, and E, so that annual CUE showed a decreasing trend and annual LUE and WUE showed increasing trends over the 19 years. The long‐term trends in the RUEs were related to the increasing CO₂ concentration. Further analysis of detrended RUEs showed that their interannual variation was impacted most strongly by air temperature. Two‐factor linear models combining CO₂ concentration and air temperature performed well (R²~0.60) in simulating annual RUEs. LUE and WUE were positively correlated both annually and seasonally, while LUE and CUE were mostly negatively correlated. Our results showed divergent long‐term trends among CUE, LUE, and WUE and highlighted the need to account for the combined effects of climatic controls and the ‘CO₂ fertilization effect’ on long‐term variations in RUEs. Since most RUE‐based models rely primarily on one resource limitation, the observed patterns of relative change among the three RUEs may have important implications for RUE‐based modeling of C fluxes.