Attention-deficit/hyperactivity disorder symptoms and stress-related biomarkers.

OBJECTIVE: The current study examined whether (a) Attention-Deficit/Hyperactivity Disorder (ADHD) symptoms were associated with dysregulation of stress-related mechanisms, and (b) whether ADHD symptoms interact with affective disorders in their association with dysregulated stress-related mechanisms. METHODS: Data were obtained from 2307 subjects participating in the Netherlands Study of Depression and Anxiety. Stress-related mechanisms were reflected by the following biomarkers: (1) hypothalamic-pituitary-adrenal axis indicators (salivary cortisol awakening curve, evening cortisol, cortisol suppression after a 0.5mg dexamethasone suppression test (DST)); (2) autonomic nervous system measures (heart rate, pre-ejection period, respiratory sinus arrhythmia); (3) inflammatory markers (C-reactive protein, interleukin-6, tumor necrosis factor-alpha); (4) brain-derived neurotrophic factor. ADHD symptoms were measured using Conners' Adult ADHD Rating Scale and used both dichotomous (High ADHD symptoms (yes/no)) and continuous (Inattentive symptoms, Hyperactive/Impulsive symptoms, and the ADHD index). RESULTS: Regression analyses showed associations between High ADHD symptoms, Inattentive symptoms, the ADHD index and a higher cortisol awakening curve, between Hyperactive/Impulsive symptoms and less cortisol suppression after DST, and between Inattentive symptoms and a longer pre-ejection period. However, the associations with the cortisol awakening curve disappeared after adjustment for depressive and anxiety disorders. No associations were observed between ADHD symptoms and inflammatory markers or BDNF. ADHD symptoms did not interact with affective disorders in dysregulation of stress-related mechanisms. CONCLUSION: Some associations were observed between ADHD symptoms, the HPA-axis, and the pre-ejection period, but these were mostly driven by depressive and anxiety disorders. This study found no evidence that ADHD symptomatology was associated with dysregulations in inflammatory markers and BDNF. Consequently, ADHD symptoms did not confer an added risk to the disturbances of stress-related mechanisms in an - already at-risk - population with affective disorders.

Attention-deficit hyperactivity disorder symptoms add risk to circadian rhythm sleep problems in depression and anxiety.

BACKGROUND: Comorbid ADHD symptoms may partly account for circadian rhythm disturbances in depression and anxiety disorders. METHODS: Self-reported sleep characteristics of 2090 participants in the Netherlands Study of Depression and Anxiety were assessed using the Munich Chronotype Questionnaire. We defined 3 groups: healthy controls (HC), persons with lifetime depression and/or anxiety disorders (LDA), and those with both LDA and high ADHD symptoms (LDA+ADHD), using the Conner's Adult ADHD Rating Scale. RESULTS: Sleep characteristics were least favorable in the LDA+ADHD group. Important group differences between LDA+ADHD, LDA and HC were found for extremely late chronotype (12% vs. 5% vs. 3%; p<.001), sleep duration <6h (15% vs. 5% vs. 4%; p<.001), and for an indication of the Delayed Sleep Phase Syndrome (DSPS; 16% vs. 8% vs. 5%; p<.001). After adjustment for covariates, including depression and anxiety, presence of ADHD symptoms increased the odds ratio for late chronotype (OR=2.6; p=.003), indication of DSPS (OR=2.4; p=.002), and sleep duration <6h (OR=2.7; p=.007). LIMITATIONS: ADHD conceptually overlaps with symptom presentation of depression and anxiety. We used a cross-sectional study design, and used self reported sleep characteristics. CONCLUSIONS: High ADHD symptoms were associated with an increased rate of circadian rhythm sleep disturbances in an already at-risk population of people with depression and/or anxiety disorders. Circadian rhythm sleep disorders, as often seen in ADHD are not entirely due to any comorbid depression and/or anxiety disorder. Adequate treatment of such sleep problems is needed and may prevent serious health conditions in the long term.

Bacterial abundance and composition in marine sediments beneath the Ross Ice Shelf, Antarctica.

Marine sediments of the Ross Sea, Antarctica, harbor microbial communities that play a significant role in the decomposition, mineralization, and recycling of organic carbon (OC). In this study, the cell densities within a 153-cm sediment core from the Ross Sea were estimated based on microbial phospholipid fatty acid (PLFA) concentrations and acridine orange direct cell counts. The resulting densities were as high as 1.7 × 107 cells mL⁻¹ in the top ten centimeters of sediments. These densities are lower than those calculated for most near-shore sites but consistent with deep-sea locations with comparable sedimentation rates. The δ¹³C measurements of PLFAs and sedimentary and dissolved carbon sources, in combination with ribosomal RNA (SSU rRNA) gene pyrosequencing, were used to infer microbial metabolic pathways. The δ¹³C values of dissolved inorganic carbon (DIC) in porewaters ranged downcore from -2.5‰ to -3.7‰, while δ¹³C values for the corresponding sedimentary particulate OC (POC) varied from -26.2‰ to -23.1‰. The δ¹³C values of PLFAs ranged between -29‰ and -35‰ throughout the sediment core, consistent with a microbial community dominated by heterotrophs. The SSU rRNA gene pyrosequencing revealed that members of this microbial community were dominated by ß-, δ-, and γ-Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Among the sequenced organisms, many appear to be related to known heterotrophs that utilize OC sources such as amino acids, oligosaccharides, and lactose, consistent with our interpretation from δ¹³CPLFA analysis. Integrating phospholipids analyses with porewater chemistry, δ¹³CDIC and δ¹³CPOC values and SSU rRNA gene sequences provides a more comprehensive understanding of microbial communities and carbon cycling in marine sediments, including those of this unique ice shelf environment.

Evaluation of 16S, map1 and pCS20 probes for detection of Cowdria and Ehrlichia species.

A panel of 16S ribosomal RNA gene probes has been developed for the study of the epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five distinct genotypes; one detects any Group III Ehrlichia species other than Cowdria and one detects any Group II Ehrlichia species. These probes have been used on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other cowdria genes (map1 and pCS20) which have previously been used for heartwater epidemiology. This paper describes the first direct comparison of all currently available DNA probes for heartwater-associated organisms.

Comparative evaluation of 16S, map1 and pCS20 probes for the detection of Cowdria and Ehrlichia species in ticks.

We have developed a panel of 16S ribosomal RNA gene probes for heartwater epidemiology; five of these detect different Cowdria genotypes (Ball3, Senegal, Omatjenne, Crystal Springs, and Mara 87/7); one detects all five of these genotypes; one detects any Group III Ehrlichia species other than Cowdria; one detects any Group II Ehrlichia species. We have used these probes on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different Cowdria isolates, field ticks were harvested from animals in heartwater-endemic and heartwater-free areas. All the samples were also examined by PCR amplification and probing for two other Cowdria genes (map1 and pCS20) which have been used for heartwater epidemiology. This paper describes the first direct comparison of all the currently available DNA probes for heartwater-associated organisms.

A partially protective clone from Cowdria ruminantium identified by using a Salmonella vaccine delivery system.

A strategy has been developed to screen the Cowdria genome, using a Salmonella vaccine delivery system, to identify genes that code for protection-stimulating proteins. We have cloned mini-libraries of Cowdria into this Salmonella system, used the recombinant bacteria to immunize outbred mice, and then challenged them after two weeks with a lethal dose of Cowdria. When one of these mini-libraries was tested in a group of 5 mice, one mouse lived much longer than the others. The experiment was repeated with each of the clones from the mini-library being tested individually in 10 mice, and one mouse survived the challenge. This clone has been tested repeatedly in larger groups of mice and is proven to protect 14% of outbred mice against a lethal Cowdria challenge.

Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences.

Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp. Sequenced difference within the V1 loop were sufficient for the derivation of four Cowdria genotype-specific oligonucleotide probes. Four further probes were designed; one for the detection of any Cowdria genotype, one for the detection of any Group II Ehrlichia sp., one for any Group III Ehrlichia sp. and one for all Pseudomonadaceae. All the probes were specific except that for the Cowdria (Ball 3) genotype. The high prevalence (96%) in field samples of pseudomonad-like 16S sequences was the result of environmental contamination. The probes were used to screen DNA from goats in an area free of both Amblyomma ticks and clinical heartwater. A substantial proportion (42%) gave positive reactions for the apparently apathogenic Cowdria (Omatjenne), indicating that this genotype is relatively common.

Development of the OPgun for bombardment of animal tissues.

A simple and inexpensive particle-bombardment device, the OPgun, was constructed for the delivery of DNA into animal tissues. This device is based on the particle-inflow gun first described for plant-cell transfection. The delivery of tungsten particles into the epidermis of the mouse ear, without the use of vacuum and without causing damage to the tissue, was demonstrated. The system was also shown to be capable of inducing antibodies to a foreign gene in mice.

In vitro isolation and cultivation of Cowdria ruminantium under serum-free culture conditions.

Three stocks of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, were propagated in bovine endothelial cells in a serum-free culture medium. The Vosloo, Welgevonden and Senegal stocks were propagated for a period of more than 203, 134, and 43 days, respectively. Two of the C ruminantium stocks (Vosloo and Senegal) were also successfully initiated under serum-free culture conditions. The serum-free medium consisted of a modified HL-1 medium. The Senegal stock was successfully propagated in Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 containing 10 per cent fetal bovine serum.

Rinderpest in South Africa--100 years ago.

To commemorate the centenary of the outbreak of rinderpest in South Africa, the historical events leading up to and following this major epidemic are recounted. Its impact on livestock and human populations, as well as initial and eventual successful attempts to control it are discussed.

Immunohistochemical identification of Cowdria ruminantium in formalin-fixed tissue sections from mice, sheep, cattle and goats.

An immunohistochemical staining technique in which a monospecific serum was used against the major antigenic protein -1 (MAP-1) of Cowdria ruminantium, was evaluated for the detection of C. ruminantium in formalin-fixed tissues of experimentally infected mice and field cases of heartwater in sheep, cattle and goats. Mice were infected with the mouse-pathogenic stocks: Mara, Kwanyanga, Welgevonden, Nonile, Vosloo, Kümm, Mali and Omatjenne. In all these cases and in the naturally infected cattle, sheep and goats, Cowdria colonies were identified as clearly-defined, brown-staining rickettsial colonies within the cytoplasm of endothelial cells. No positive staining was observed in the control group. This technique was shown to be reliable for detecting infection with C. ruminantium in the formalin-fixed tissues of mice and domestic ruminants.

Vaccination against heartwater using in vitro attenuated Cowdria ruminantium organisms.

Sequential passage of Cowdria ruminantium (Senegal isolate) in cultures of bovine umbilical endothelial cells has resulted in loss of virulence without loss of immunogenicity, as previously demonstrated. We have carried out further immunization of 39 Dutch sheep using in vitro attenuated rickettsiae of passage 21 and challenged these animals either with the homologous or with heterologous Cowdria stocks. After vaccination several sheep developed elevated rectal temperatures for a maximum of 2 days, but no further clinical response to the vaccine was observed. All sheep developed high titres of antibodies to Cowdria. Challenge of 10 sheep with the homologous virulent stock did not provoke any clinical reaction, demonstrating that these animals were solidly immune. Reactions to heterologous challenge varied from virtually no reaction to fatal heartwater depending on the stock of Cowdria used. These results are discussed in relation to currently available vaccination methods against cowdriosis. In Senegal 30 susceptible sahelian sheep were immunized with attenuated rickettsiae of passage 21. Hyperthermia was seen in 13, the only other clinical symptom was a temporary diarrhoea. The immunized animals are at present exposed, together with 30 controls, to field challenge in the Niayes, the area where the Senegal isolate was originally isolated.