Routes of intraspecies transmission of Mycobacterium avium subsp. paratuberculosis in rabbits (Oryctolagus cuniculus): a field study

Rabbits have been increasingly linked to the persistence of paratuberculosis (Johne's disease) in domestic ruminants in the United Kingdom. The aims of this study were to determine the routes of intraspecies transmission of Mycobacterium avium subspecies paratuberculosis (MAP) in rabbits and to estimate the probability of transmission via each route, in order to gain understanding of the dynamics of MAP in this host. Rabbits were sampled from two sites where MAP had previously been isolated from the livestock and rabbit populations. No pathology was noted in any animals, but the overall prevalence of MAP in rabbits was high at both sites studied, 39.7% and 23.0%, respectively. MAP was isolated from the testes, uterus, placenta, fetuses, and milk. This is the first time that the bacterium has been isolated from any of these tissues in a nonruminant wildlife species. These results suggest that transmission may occur vertically, pseudovertically, and horizontally. Vertical, i.e., transplacental, and/or pseudo-vertical, i.e., through the ingestion of contaminated milk and/or feces, transmission occurred in 14% of offspring entering the population at 1 month of age. As infection via these routes is only possible from infected adult females, this equates to a probability of infection via this route of 0.326. Probability of infection via horizontal transmission (including interspecies transmission) occurred at up to 0.037 per month. The presence of these routes of transmission within natural rabbit populations will contribute to the maintenance of MAP infections within such populations and, therefore, the environment.     

Prenatal development of cutaneous afferent connections in the spinal cord of fetal sheep

In this study we have examined the physiological and neurochemical development of the cutaneous afferent pathways from the hindlimb to the spinal cord in fetal sheep. We have shown that somatosensory input from the hindlimb evokes activity in DRG neurons at 87d gestation and in cells in the dorsal horn at 92d (term, 146d). There is evidence of immunoreactivity for substance P, calcitonin gene-related peptide and glutamine several days prior to this at 77-80 days. The implication of these findings are discussed.     

Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae

In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22 000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir.     

Pulmonary and systemic endotoxin tolerance in preterm fetal sheep exposed to chorioamnionitis

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term=150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.     

Protective antifungal effect of neem (Azadirachta indica) extracts on mango (Mangifera indica) and rain tree (Albizia saman) wood

Neem (Azadirachta indica) extract (NE) and NE combined with copper sulfate and boric acid (NECB) were examined for their protective effect against fungal deterioration of mango (Mangifera indica) and rain tree (Albizia saman) wood. Growth of the white-rot fungus Schizophyllum commune was completely inhibited on solid medium containing 1.8% (w/w) NE or 5% (w/w) NECB. The average weight losses of NE and NECB treated wood blocks inoculated with S. commune were respectively 4.7% and 3.3% for M. indica and 4.1% and 3.0% for A. saman. These numbers were significantly lower compared to those obtained in the untreated condition. Similar observations were also noticed in the case of field tests. An average increase of life span of M. indica and A. saman due to NE treatment compared to untreated samples was about 6–7 times higher. Therefore, both NE and NECB treatments are promising preservation options for enhancing the durability of both M. indica and A. saman woods.     

Urine Selenium Changes During Pregnancy Do Not Correlate with Thyroid Autoantibodies in a Mildly Iodine Deficient Population

Selenium (Se) is a key component of iodinases; higher Se levels are associated with lower titers of antithyroid peroxidase antibodies (anti-TPO). Pregnancy exerts profound effects on thyroid function and autoimmunity. To assess the relationship of urine Se levels with thyroid function and autoimmunity in pregnant women residing in Athens, Greece, we studied prospectively 47 euthyroid women in uncomplicated singleton pregnancies (mean age + SD: 30?+?5 years) in each trimester, measuring urine Se levels, urine iodine, plasma thyrotropin (TSH), free thyroxine and triiodothyronine (FT4 and FT3), as well as levels of anti-TPO antibodies. Changes of the measured parameters were assessed over each trimester; thyroid parameters were assessed with relation to Se levels. Urine Se dropped by the third trimester, whereas urine iodine did not change appreciably during pregnancy. TSH and anti-TPO did not show appreciable changes; FT4 and FT3 gradually decreased as the pregnancy advanced. No relationship between urine Se levels and anti-TPO was found. During pregnancy, changes in urine Se levels accompany mild changes in thyroid function. However, we did not find some association between these changes and thyroid autoimmune activity over this period, probably because the effect of Se on thyroid autoimmunity may only become apparent in case of excess Se fortification.     

High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention

CD8(+) tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-γ production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase Cγ) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (IκB, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8(+) T cells infiltrating non-tumor kidney areas (CD8(+) non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-α (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8(+) T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy.