Molecular cloning and expression profiles of calreticulin gene from the diamondback moth,Plutella xylostella

Calreticulin (CRT) plays pivotal roles in Ca²⁺ homeostasis, molecular chaperoning, infection, inflammation and innate immunity. In an attempt to study the involvement of CRT in innate immunity, the full-length cDNA of calreticulin (PxCRT) was cloned from the diamondback moth, Plutella xylostella. It consists of 1674 bp (excluding poly-A tail) with a longest open reading frame (ORF) of 1197 bp encoding 398 amino acids. In silico analysis of PxCRT ORF reveals that it has various repeat motifs and endoplasmic reticulum retention signal found in all the calreticulin proteins. As expected, high amino acid sequence identities were found from other CRTs identified from Bombyx mori (87%), Galleria mellonella (87%), Apis mellifera (74%), Anopheles gambiae (74%), Tribolium castaneum (73%), Culex quinquefasciatus (73%), Rhodnius prolixus (72%), Nasonia vitripennis (71%), Drosophila melanogaster (71%) and Haemaphysalis qinghaiensis (68%). During development, P. xylostella expressed PxCRT predominantly in the pupal stage. In addition, spatial expression pattern analysis indicates that PxCRT was highly expressed in the silk gland. PxCRT mRNA, furthermore, was strongly induced 3 to 6 h after laminarin treatment, suggesting that PxCRT appears to be involved in immune responses and also plays an important role in the silk gland.     

Hemin interactions and alterations of the subcellular localization of prion protein

Hemin (iron protoporphyrin IX) is a crucial component of many physiological processes acting either as a prosthetic group or as an intracellular messenger. Some unnatural, synthetic porphyrins have potent anti-scrapie activity and can interact with normal prion protein (PrPC). These observations raised the possibility that hemin, as a natural porphyrin, is a physiological ligand for PrPC. Accordingly, we evaluated PrPC interactions with hemin. When hemin (3-10 microM) was added to the medium of cultured cells, clusters of PrPC formed on the cell surface, and the detergent solubility of PrPC decreased. The addition of hemin also induced PrPC internalization and turnover. The ability of hemin to bind directly to PrPC was demonstrated by hemin-agarose affinity chromatography and UV-visible spectroscopy. Multiple hemin molecules bound primarily to the N-terminal third of PrPC, with reduced binding to PrPC lacking residues 34-94. These hemin-PrPC interactions suggest that PrPC may participate in hemin homeostasis, sensing, and/or uptake and that hemin might affect PrPC functions.     

Effect of hookworm infection on wheat challenge in celiac disease--a randomised double-blinded placebo controlled trial

Background and Aims

The association between hygiene and prevalence of autoimmune disease has been attributed in part to enteric helminth infection. A pilot study of experimental infection with the hookworm Necator americanus was undertaken among a group of otherwise healthy people with celiac disease to test the potential of the helminth to suppress the immunopathology induced by gluten.

Methods

In a 21-week, double-blinded, placebo-controlled study, we explored the effects of N. americanus infection in 20 healthy, helminth-naïve adults with celiac disease well controlled by diet. Staged cutaneous inoculations with 10 and 5 infective 3^rd stage hookworm larvae or placebo were performed at week-0 and -12 respectively. At week-20, a five day oral wheat challenge equivalent to 16 grams of gluten per day was undertaken. Primary outcomes included duodenal Marsh score and quantification of the immunodominant α-gliadin peptide (QE65)-specific systemic interferon-γ-producing cells by ELISpot pre- and post-wheat challenge.

Results

Enteric colonisation with hookworm established in all 10 cases, resulting in transiently painful enteritis in 5. Chronic infection was asymptomatic, with no effect on hemoglobin levels. Although some duodenal eosinophilia was apparent, hookworm-infected mucosa retained a healthy appearance. In both groups, wheat challenge caused deterioration in both primary and several secondary outcomes.

Conclusions

Experimental N. americanus infection proved to be safe and enabled testing its effect on a range of measures of the human autoimmune response. Infection imposed no obvious benefit on pathology.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00671138","term_id":"NCT00671138"}}NCT00671138     

Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis

An overview of the morphology and life cycle of Batrachochytrium dendrobatidis, the cause of chytridiomycosis of amphibians, is presented. We used a range of methods to examine stages of the life cycle in culture and in frog skin, and to assess ultrastructural pathology in the skin of 2 frogs. Methods included light microscopy, transmission electron microscopy with conventional methods as well as high pressure freezing and freeze substitution, and scanning electron microscopy with critical point drying as well as examination of bulk-frozen and freeze-fractured material. Although chytridiomycosis is an emerging disease, B. dendrobatidis has adaptations that suggest it has long been evolved to live within cells in the dynamic tissue of the stratified epidermis. Sporangia developed at a rate that coincided with the maturation of the cell, and fungal discharge tubes usually opened onto the distal surface of epidermal cells of the stratum corneum. A zone of condensed, fibrillar, host cytoplasm surrounded some sporangia. Hyperkeratosis may be due to (1) a hyperplastic response that leads to an increased turnover of epidermal cells, and (2) premature keratinization and death of infected cells.     

Fixation of mucus on rainbow trout (Oncorhynchus mykiss Walbaum) gills for light and electron microscopy

Fixation of mucus for the assessment of biofilms and surface associated pathogens often involves complex and expensive techniques. Rainbow trout killed by an overdose of MS 222 had their gills removed and immersion-fixed gently in buffered glutaraldehyde containing 2% Alcian blue. Control tissues consisted of gills fixed in Alcian blue-free fixative. Trout were also killed and directly immersed in liquid nitrogen and the gills freeze-dried then vapour fixed with osmium tetroxide at −50° C. Following fixation gill tissue was processed for light and electron microscopy. A continuous and intact mucous coat was not detected on tissue fixed by conventional methods but the addition of Alcian blue to the fixative preserved an extensive mucous coat trapped between the lamellae and overlying the epithelia. Electron microscopic examination revealed that mucus preservation with the conventional fixative was poor and intermittent whereas the addition of Alcian blue to the fixative greatly enhanced the preservation of the branchial mucous coat. Mucus appeared as interdispersed flocculant material between the epithelial microridges and formed extensive superficial sheets over the epithelium. Freeze-dried/vapour-fixed gill tissue also provides excellent preservation of the integrity of branchial mucous coats, the mucus appearing as a continuous sheet over the filament and secondary lamellae. However, freeze-dried tissue fails to preserve sufficient cellular integrity for this technique to be useful for light or transmission electron microscopy. The potential for use of glutaraldehyde-Alcian blue fixed-gill tissue diagnostically and in research are discussed.     

The Decline of the Sharp-Snouted Day Frog (Taudactylus acutirostris): The First Documented Case of Extinction by Infection in a Free-Ranging Wildlife Species?

Infectious diseases are increasingly recognized as the cause of mass mortality events, population declines, and the local extirpation of wildlife species. In a number of cases, it has been hypothesized that pathogens have caused species extinctions in wildlife. However, there is only one definitively proven case of extinction by infection, and this was in a remnant captive population of a Polynesian tree snail. In this article, we review the potential involvement of infectious disease in the recent extinction of the sharp-snouted day frog Taudactylus acutirostris. Our review of available evidence suggests that a virulent pathogen of amphibians, Batrachochytrium dendrobatidis, caused a rapid, catastrophic decline of this species, from which it did not recover. We propose that this is the first case of extinction by infection of a free-ranging wildlife species where disease acted as both the proximate and ultimate cause of extinction. This highlights a probable underreporting of infectious disease as a cause of biodiversity loss historically and currently.     

Induction time for resistance to microsporidial gill disease caused by Loma salmonae following vaccination of rainbow trout (Oncorhynchus mykiss) with a spore-based vaccine

Resistance to re-infection of rainbow trout to Loma salmonae, a microsporidian gill parasite has been previously documented and this study examined how rapidly this resistance develops. Naive rainbow trout were inoculated intraperitoneally (IP) with an inactivated spore-based vaccine and were then given an oral challenge with a high dose of L. salmonae spores at various weeks after being vaccinated. Non-vaccinated naive fish (exposed group) were challenged alongside of each group of vaccinated fish to ensure that the challenges were relatively standardised. In each group of fish, four weeks after the challenge, numbers of xenomas were counted on a gill arch for all fish. Vaccinated trout were completely resistant to a L. salmonae challenge six weeks after vaccination, although the onset of resistance began at approximately week 3, as observed with a reduction in the percent infected and xenoma intensity. The maximum percent infected for the vaccinated fish was 83% following a challenge two weeks following vaccination, whereas for the exposed group the maximum prevalence of 100% was reached several times. With continued research, a spore-based vaccine for L. salmonae has the potential to become the first commercially available parasite vaccine for fish.