Rapid-bTB: a novel lateral flow test able to differentiate Mycobacterium bovis from Mycobacterium tuberculosis in sputum

Background: A novel lateral flow, immunochromatographic assay (LFD) specific for Mycobacterium bovis, the cause of bovine tuberculosis and zoonotic TB, was recently developed at Queen’s University Belfast. The LFD detects whole M. bovis cells, in contrast to other commercially available LFD tests (BD MGITTM TBc ID, SD Bioline TB Ag MPT 64, Capilia TB-Neo kit) which detect MPT64 antigen secreted during growth. The new LFD test has been evaluated in the veterinary context, and its specificity for M. bovis in the broadest sense (i.e. subsp. bovis, subsp. caprae and BCG) and sensitivity to detect M. bovis in positive MGIT™ liquid cultures was demonstrated comprehensively.
Methods: Preliminary work was carried out by researchers at Queen’s University Belfast to optimise sputum sample preparation, estimate the limit of detection (LOD) of the LFD with M. bovis-spiked sputum samples, and check LFD specificity by testing a broad range of non-tuberculous Mycobacterium spp. (NTM) and other bacterial genera commonly encountered in sputum samples (Haemophilus, Klebsiella, Pseudomonas, Staphylococcus). In the Cameroon laboratory direct detection of M. bovis in human sputa was attempted, and 50 positive sputum MGIT™ cultures and 33 cultures of various Mycobacterium spp. originally isolated from human sputa were tested.
Results: Sputum sample preparation consisted of digestion with 1% NALC for 30 min, centrifugation at 3000g for 20 min, PBS wash, centrifugation again, and pellet resuspended in KPL blocking buffer before 100 µl was applied to the LFD. The LOD of the LFD applied to M. bovis-spiked sputum was estimated to be 104 CFU/ml. A small number of confirmed Ziehl-Neelsen ‘3+’ M. bovis positive sputum samples were tested directly but no positive LFD results were obtained. All of the sputum MGIT™ cultures and mycobacterial cultures (including M. tuberculosis, M. africanum, M. bovis, M. intracellulare, M. scrofulaceum, M. fortuitum, M. peregrinum, M. interjectum) tested LFD negative when read after 15 min except for the M. bovis cultures, thereby confirming specificity of LFD for M. bovis in the clinical microbiology context.
Conclusions: Results indicate that the ‘Rapid-bTB’ LFD is a very specific test, able to differentiate M. bovis from M. tuberculosis, M. africanum, and a range of NTM isolated from human sputa in MGITTM liquid cultures. However, the LFD lacks sufficient sensitivity to be applied earlier in the diagnostic process to directly test human sputa.

The differential effects of chlorpromazine and haloperidol on latent inhibition in healthy volunteers.

Latent inhibition (LI) is a measure of reduced learning about a stimulus to which there has been prior exposure without any consequence. It therefore requires a comparison between a pre-exposed (PE) and a non-pre-exposed (NPE) condition. Since, in animals, LI is disrupted by amphetamines and enhanced by antipsychotics, LI disruption has been proposed as a measure of the characteristic attentional deficit in schizophrenia: the inability to ignore irrelevant stimuli. The findings in humans are, however, inconsistent. In particular, a recent investigation suggested that since haloperidol disrupted LI in healthy volunteers, and LI was normal in non-medicated patients with schizophrenia, the previous findings in schizophrenic patients were entirely due to the negative effects of their medication on LI (Williams et al., 1998). We conducted two studies of antipsychotic drug effects on auditory LI using a within-subject, parallel group design in healthy volunteers. In the first of these, single doses of haloperidol (1 mg. i.v.) were compared with paroxetine (20 mg p.o.) and placebo, and in the second, chlorpromazine (100 mg p.o.) was compared with lorazepam (2 mg. p.o.) and placebo. Eye movements, neuropsychological test performance (spatial working memory (SWM), Tower of London and intra/extra dimensional shift, from the CANTAB test battery) and visual analogue rating scales, were also included as other measures of attention and frontal lobe function. Haloperidol was associated with a non-significant reduction in LI scores, and dysphoria/akathisia (Barnes Akathisia Rating Scale) in three-quarters of the subjects. The LI finding may be explained by increased distractibility which was indicated by an increase in antisaccade directional errors in this group. In contrast, LI was significantly increased by chlorpromazine but not by an equally sedative dose of lorazepam (both drugs causing marked decreases in peak saccadic velocity). Paroxetine had no effect on LI, eye movements or CANTAB neuropsychological test performance. Haloperidol was associated with impaired SWM, which correlated with the degree of dysphoria/akathisia, but no other drug effects on CANTAB measures were detected. We conclude that the effect of antipsychotics on LI is both modality and pharmacologically dependent and that further research using a wider range of antipsychotic compounds is necessary to clarify the cognitive effects of these drugs, and to determine whether there are important differences between them.

Pre-treatment with Depo-Provera modifies the pharmacokinetics of CMPD167 in rhesus macaques following vaginal ring administration

BACKGROUND: Vaginal ring devices are being developed to provide sustained release of HIV microbicides. To date, only limited pharmacokinetic data is available from animal or human studies. Here we report the effect of Depo-Provera (DP) pre- treatment, commonly used to thin the vaginal epithelium in challenge experiments, on the pharmacokinetic profile of CMPD167 (a small molecule CCR5 co-receptor antagonist) in rhesus macaques following vaginal ring administration.

METHODS: A single 400mg CMPD167 silicone elastomer vaginal ring was inserted into each of twelve female rhesus macaques. Six macaques were treated with (DP) 30 days before ring placement; the other six macaques were untreated. Blood, vaginal fluid and vaginal biopsies were collected prior to and at various times during 28 days of ring placement and assayed for CMPD167 levels by HPLC. Rings were assayed for residual CMPD167 at the end of the study and the calculated amount of CMPD167 released in vivo compared with in vitro release data.

RESULTS: Vaginal fluid, plasma and tissue levels of CMPD167 were detectable throughout ring placement. Significant differences were observed in mean daily vaginal fluid levels between the DP-treated (16–56 mcg/mL) and untreated groups (48–181 mcg/mL). Plasma CMPD167 levels were significantly higher peaking at 4 ng/mL and maintaining levels of 1–2 nM throughout the 14 days of testing in animals pre-treated with DP compared to non DP-treated macaques (<1 ng/mL maintained). Tissue levels were varied between 2–10 g/mL CMPD167 with no significant difference between the DP-treated and untreated macaques.

CONCLUSIONS: The study demonstrates that clinically relevant, and possibly protective doses of CMPD167 are released in the vaginal vault of rhesus macaques from vaginal rings through 28 days duration. DP is known to induce vaginal epithelial thinning and lower vaginal fluid levels, which accounts for the increased plasma levels of CMPD167. In contrast, macaques not treated with DP had minimal absorption into plasma compartments and significantly higher levels of CMPD167 in the vagina, similar to those previously shown to be protective against vaginal challenge.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

No evidence of morphine analgesia to noxious shock in the shore crab, Carcinus maenas

A number of criteria have been suggested for testing if pain occurs in animals, and these include an analgesic effect of opiates (Bateson, 1991). Morphine reduces responses to noxious stimuli in crustaceans but also reduces responsiveness in a non-pain context. Here we use a paradigm in which shore crabs receive a shock in a preferred dark shelter but not if they remain in an unpreferred light area. Analgesia should thus enhance movement to the preferred dark area because they should not experience 'pain'. However, morphine inhibits rather than enhances this movement even when no shock is given. Morphine produces a general effect of non-responsiveness rather than a specific analgesic effect and this could also explain previous studies claiming analgesia. However, we question the utility of this criterion of pain and suggest instead that behavioural criteria be employed. (C) 2011 Published by Elsevier B.V.

RESIDUES OF CLENBUTEROL IN CATTLE RECEIVING THERAPEUTIC DOSES - IMPLICATIONS FOR DIFFERENTIATING BETWEEN LEGAL AND ILLEGAL USE

Clenbuterol (CBL) can be used legally in the treatment of respiratory diseases and illegally as a growth promoter in animals, Liver and eye have previously been shown to be effective matrices for the detection of residual concentrations of the drug.

A review of scientific literature on inherited disorders in domestic horse breeds

The full extent to which inherited disorders occur in different breeds of domestic horse (Equus caballus) has not been previously been investigated. A systematic search was carried out to review scientific literature on inherited disorders in domestic horse breeds and examine patterns in potentially inherited disorders. A two-part search was conducted: (i) electronic bibliographic databases for published studies; and (ii) existing online databases of inherited disorders in animals. A total of 230 papers were identified, discussing 102 inherited disorders in the horse. Few cases (17) were found in which disorders were reported to have a direct link to a conformational or phenotypic trait. Forty-nine breeds of domestic horse were described as being predisposed to one or more inherited disorders, but such predispositions did not distinguish between genetic or environmental causes. There were few patterns in the number of disorders to which breeds were reportedly predisposed or in the extent to which disorders were researched. The structure and grouping of disorders presented here could assist with standardisation in the terminology used for describing inherited disorders. © 2012 Universities Federation for Animal Welfare The Old School.

Dietary exposure to aflatoxin from maize and groundnut in young children from Benin and Togo, West Africa

Aflatoxins are a family of fungal toxins that are carcinogenic to man and cause immunosuppression, cancer and growth reduction in animals. We conducted a cross-sectional study among 480 children (age 9 months to 5 years) across 4 agroecological zones (SS, NGS, SGS and CS) in Benin and Togo to identify the effect of aflatoxin exposure on child growth and assess the pattern of exposure. Prior reports on this study [Gong, Y.Y., Cardwell, K., Hounsa, A., Egal, S., Turner, Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20-21, Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K., Wild, C.P., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning and weaning foods. International Journal of Epidemiology, 32, 556-562] showed that aflatoxin exposure among these children is widespread (99%) and that growth faltering is associated with high blood aflatoxinalbumin adducts (AF-alb adducts), a measure of recent past exposure. The present report demonstrates that consumption of maize is an important source of aflatoxin exposure for the survey population. Higher AF-alb adducts were correlated with higher A. flavus (CFU) infestation of maize (p=0.006), higher aflatoxin contamination (ppb) of maize (p<0.0001) and higher consumption frequencies of maize (p=0.053). The likelihood of aflatoxin exposure from maize was particularly high in agro-ecological zones where the frequency of maize consumption (SGS and CS), the presence of allatoxin in maize (SGS) or the presence of A. flavus on maize (NGS and SGS) was relatively high. Socio-economic background did not affect the presence of A. flavus and aflatoxin in maize, but better maternal education was associated with lower frequencies of maize consumption among children from the northernmost agro-ecological zone (SS) (p=0.001). The impact of groundinit consumption on aflatoxin exposure was limited in this population. High AF-alb adduct levels were correlated with high prevalence of A. flavus and aflatoxin in groundinit, but significance was weak after adjustment for weaning status, agro-ecological zone and maternal socio-economic status (resp. p = 0.091 and p = 0.083). Ingestion of A. flavus and aflatoxin was high in certain agro-ecological zones (SS and SGS) and among the higher socio-economic strata due to higher frequencies of groundnut consumption. Contamination of groundnuts was similar across socio-economic and agroecological boundaries.

In conclusion, dietary exposure to aflatoxin from groundnut was less than from maize in young children from Benin and Togo. Intervention strategies that aim to reduce dietary exposure in this population need to focus on maize consumption in particular, but they should not ignore consumption of groundnuts. (c) 2005 Elsevier B.V. All rights reserved.